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| United States Patent |
6,218,167
|
|
Allen
, et al.
|
April 17, 2001
|
Stable biocatalysts for ester hydrolysis
Abstract
The instant invention encompasses isolated stable esterase enzymes
characterized by the ability to remain stable at certain temperatures,
substrate specificities, and activity profile; the expression vectors
which can express, nucleic acids which encode for, and corresponding
protein amino acid sequence of such proteins.
| Inventors:
|
Allen; Larry (Northfield, IL);
Aikens; John (LaGrange Park, IL);
DeMirjian; David (Chicago, IL);
Vonstein; Veronika (Chicago, IL);
Fonstein; Michael (Chicago, IL);
Casadaban; Malcolm (Chicago, IL)
|
| Assignee:
|
ThermoGen, Inc. (Chicago, IL)
|
| Appl. No.:
|
058260 |
| Filed:
|
April 10, 1998 |
| Current U.S. Class: |
435/252.3; 435/196; 435/252.33; 435/320.1; 536/23.2 |
| Intern'l Class: |
C12N 001/20; C12N 009/16; C12N 015/00; C07H 021/04 |
| Field of Search: |
435/196,320.1,252.33,252.3
536/23.2
|
References Cited
Assistant Examiner: Monshipouri; M.
Attorney, Agent or Firm: McDonnell Boehnen Hulbert & Berghoff
Goverment Interests
STATEMENT OF GOVERNMENT RIGHTS
The work disclosed in this application was supported in part by Grant
Number: NCI 1-R43-CA63876-01 from the NIH-SBIR to ThermoGen Inc.,
therefore, the U.S. Government may have some rights in the present
invention.
Parent Case Text
This application is a continuation-in-part of U.S. Ser. No. 08/827,810
filed Apr. 11, 1997 (abandoned) which is a continuation-in-part of U.S.
Ser. No. 08/781,802 filed Jan. 10, 1997 (now U.S. Pat. No. 5,969,121)
which is a continuation-in-part of U.S. Ser. No. 08/694,078 filed Aug. 8,
1996 (pending) which claims priority to U.S. Ser. No. 60/019,580 filed
Jun. 12, 1996 and U.S. Ser. No. 60/009,704 filed Jan. 11, 1996.
Claims
We claim:
1. An isolated nucleic acid segment comprising the nucleic acid sequence of
FIG. 6O (E020) (SEQ ID NO. 29).
2. An isolated nucleic acid segment comprising the nucleic acid sequence of
an open reading frame encoded for by the nucleic acid of claim 1.
3. An expression vector nucleic acid construct comprising an expressible
nucleic acid which is a nucleic acid of claim 2.
4. A host cell transformed with the expression vector construct of claim 3.
Description
FIELD OF THE INVENTION
The instant disclosure is directed to the field of isolated stable
biocatalysts that are suitable for enzymatic application in commercial
pharmaceutical and chemical synthesis, DNA vectors for the production of
recombinant ester hydrolyzing proteins, host cells transformed by such
vectors, and recombinant ester hydrolyzing proteins produced by such
vectors and transformed cells.
BACKGROUND OF THE INVENTION
Esterases and Lipases.
Esterases and lipases catalyze the hydrolysis of ester bonds to produce
alcohols and carboxylic acids as shown below.
##STR1##
Esterases and lipases can be characterized by different substrate
specificities, R group or chain length preference, and unique inhibitors
(1, 2). The many esterases and lipases range from hydrolases such as the
broad carboxyl esterases which preferentially hydrolyze esters with long
carbon chain R groups, to choline esterases, and to acetyl esterases which
act on very specific substrates. In many cases, these hydrolases are also
known to show stereo- and regio-selective preferences resulting from the
chiral nature inherent in protein active sites. This preferential
hydrolytic activity make them useful for reactions requiring different
regioselectivity and stereoselectivity or for kinetic resolution methods
on racemic mixtures. For enzymes that demonstrate stereoselectivity, if R*
is a racemic mixture, the product of enzyme catalyzed hydrolysis, R.sub.1,
would be the most rapidly hydrolyzed stereoisomer while the remaining
ester designated R*' would be the enriched antipode mixed with any
remaining R.sub.1. The products can then be separated by chromatography to
provide pure R.sub.1. The availability of a large pool of esterases and
lipases with varying specificities would be useful for screening the
enzymes for specific reactions, and developing optimal protocols for
specific chemical synthesis. The expedience of this process would
facilitate the production scale-up of many useful pharmaceutical products.
In aqueous solvent systems, esterases and lipases carry out their natural
reactions: the hydrolysis of ester bonds. In vitro, these enzymes can be
used to carry out reactions on a wide variety of substrates, including
esters containing cyclic and acyclic alcohols, mono- and di-esters, and
lactams (3). By carrying out the reactions in organic solvents (4, 5)
where water is excluded, the reactions of esterases and lipases can be
reversed. These enzymes can catalyze esterification or acylation reactions
to form ester bonds (3, 6, 7). This process can also be used in the
transesterification of esters and in ring closure or opening reactions.
Optically pure chiral pharmaceuticals.
Currently, the majority of synthetic chiral pharmaceuticals are sold as
racemic mixtures. However, due to advances in the synthesis of optically
pure (single isomer) chiral compounds, this situation is changing (7).
Racemic drugs often contain one isomer which is therapeutically active and
the other enantiomer which is at best inactive and at worst a major cause
of potentially harmful side effects. The non-useful isomer in a racemic
drug is increasingly being viewed as a contaminant. Indeed, the FDA's
Policy Statement for the Development of New Drugs recommends "that the
pharmacokinetic profile of each isomer should be characterized in animals
and later compared to the clinical pharmacokinetic profile obtained in
Phase I" drug testing (8). Thus, pharmaceutical companies will need to
develop a synthesis or separation route to produce each pure isomer of
each new synthetic drug.
Enzymatic synthesis of optically pure pharmaceuticals and intermediates.
Since it is often very difficult to generate optically pure solutions of
certain chiral molecules by classical chemical synthesis, new enzymatic
biocatalysts will play a major role in this endeavor. In some cases,
enzymes may be able to replace hazardous chemical synthesis procedures
with more environmentally-friendly biological synthesis processes. It can
also be much more cost effective to produce a pharmaceutical intermediate
enzymatically if an enzyme can eliminate several chemical protection and
deprotection steps at once (7). All six major classes of enzymes
(oxidoreductases, transferases, hydrolases, lyases, isomerases, and
ligases) have been useful in the synthesis of optically pure compounds as
described in several detailed reviews (3, 7). The hydrolases have proven
to be the most useful group of enzymes, due to the abundance of
hydrolases, the information about them, their independence from cofactors,
and the wide variety of substrates they can accept.
A survey of the literature shows many examples of mesophilic hydrolases
particularly esterases and lipases used in chemical synthesis or chiral
resolution. These include esterases from pig (9, 10) and horse (3) livers
and a wide variety of lipases from Aspergillus sp. (11) Candida sp.
(12-16), Pseudomonas sp., (17-19), Rhizopus sp. (20) and others. Several
lipases have been used in the synthesis of propranolol (7), a
beta-adrenergic blocking agent used in the treatment of angina and
hypertension. Ibuprofen, a nonstearoidal antiinflammatory agent has been
synthesized via stereo selective hydrolysis of its methyl ester using
carboxyesterase (7). While these enzymes have begun to demonstrate the
utility of biocatalysts in chemical synthesis, there is still a profound
need for a wider variety of esterases and lipases which have varying
substrate specificities, regioselectivities, and steroselectivities. In
addition, since these enzymes need to be employed in a large-scale
industrial setting, there is a need for them to have increased stability,
higher thermotolerance and a longer "shelf life".
Thermostable enzymes.
Thermophilic organisms have already provided a rich source of useful
proteins that catalyze reactions at higher temperatures and are stable for
much longer periods of time (21, 22). One example is the DNA Polymerase I
from Thermus aquaticus and its use in polymerase chain reaction (PCR) (23,
24). Thermophilic enzymes have become the most commercially successful
enzymes in industry because of their long-term stability and ease of use.
The most successful enzyme to date, alpha-amylase, is used in corn
processing and comes from the moderate thermophile B. stearothermophilus
(25). Another commercially successful industrial enzyme is subtilisin, a
serine protease also found in various strains of Bacillus, has been widely
used in laundry detergents and other cleaning solutions.
The commercial success of these enzymes can be attributed to their ease of
use. In addition to functioning at high temperatures, thermostable enzymes
generally posses an increased shelf life which markedly improves handling
conditions, especially by those not trained in biochemistry to work with
the specific range of conditions used for mesophilic enzymes. If enzymes
are to play a significant role in large scale processing of chemicals,
they must be able to endure the harsh conditions associated with these
processes. Thermostable enzymes are easier to handle, last longer, and
given the proper immobilization support should be reusable for multiple
applications
Finally, the hydrophobic and electrostatic forces that allow these enzymes
to survive high temperatures also allow them to generally function better
in organic solvents (26-31). While most enzymes lose a significant portion
of their activity in organic solvents, thermostable enzymes may prove more
tolerant to the denaturing conditions of many organic solvents. Highly
thermostable esterases and lipases are necessary to expand the application
of these biocatalysts in large scale industrial reactions.
Thermostable esterases and lipases.
To date, only one esterase and a few lipases have been reported with
moderately thermostable characteristics. Tulin et al. (32) reported a
Bacillus stearothermophilus esterase cloned into Bacillus brevis which was
stable up to 10 minutes at 70.degree. C. Sugihara et al.(33, 34) have
isolated novel thermostable lipases from two microorganisms, A Bacillus
soil isolate and a Pseudomonas cepacia soil isolate. The former lipase is
stable up to 30 minutes at 65.degree. C. but rapidly inactivated above
this temperature. The lipase from Pseudomonas cepacia was stable when
heated for 30 minutes at 75.degree. C. and pH 6.5 but had only 10% of its
activity when assayed at this temperature. A thermoalcalophilic lipase
(35) was identified from a Bacillus species MC7 isolated by continuous
culture and had a half-life of 3 hours at 70.degree. C. Finally,
Sigurgisladottir et al. (6) have reported the isolation of one Thermus and
two Bacillus strains which posses lipases active on olive oil up to
80.degree. C., although there was no report on enzyme stability in this
study.
These enzymes offer only limited variations in substrate specificities and
only moderate thermostability profiles. They do not address the need for
different substrate specificities, the need to produce large scale
quantities which can be economically commercialized, and many of them have
only limited overall stability. In this patent application we have
identified a series of esterases and lipases which offer a range of
substrate specificities (including regioselectivity, stereoselectivity),
enhanced enzyme stability, and can be produced in large quantities for
commercial use.
SUMMARY OF THE INVENTION
The instant invention provides for the isolation and characterization of
commercial grade enzyme preparations characterized by esterase activity,
and corresponding to the data as disclosed in Table 1. In a preferred
embodiment, the instant invention provides for the isolation, and
characterization of specifically purified esterase which is characterized
by esterase activity, and corresponding to the data as disclosed in Table
1. In a most preferred embodiment, the instant invention provides for
proteins generated by recombinant DNA technology which have esterase
activity. The instant invention encompasses lambda phage expression
vectors which contain an insert that can be used for the production of
recombinant ester hydrolyzing proteins of the instant invention, from a
transformed cell host. The insert contained on the lambda phage expression
vector may be used in, for example, a phage-plasmid hybrid expression
vector or other suitable expression vector such as, but not limited to,
plasmids, YACs, cosmids, phagemids, etc. In a preferred embodiment, a
lambda expression vector is one of the vectors named in Table 7, or one
which contains an insert which encodes for a substantially similar
recombinant protein. The instant disclosure also provides for vectors
which are capable of transforming a host cell, and which encode for
recombinant ester hydrolyzing proteins, the transformed host cells, and
the recombinant ester hydrolyzing protein. Appropriate host cells include
but are not limited to: E. coli, Bacilli, Thermus sp., etc. The
recombinant ester hydrolyzing protein encoded by the vector is capable of
hydrolyzing 5-bromo-4-chloro-3-indolyl-acetate (X-acetate). The
recombinant ester hydrolyzing protein produced by the vector can be
further characterized by a half-life stability comparable to that of a
corresponding protein purified from the isolates. The recombinant ester
hydrolyzing protein is also characterized by the ability to remain stable
at temperatures comparable to, or better than that of the corresponding
protein from the original isolates. Recombinant ester hydrolyzing protein
encoded for by the vector can also be characterized by certain substrate
specificities as discussed below, which are comparable to those of the
corresponding purified protein from the isolates. In a preferred
embodiment the vector is a vector named in Table 7 or 8, or one which
contains an insert which encodes for a substantially similar recombinant
protein. In a preferred embodiment of the instant invention, a vector
which encodes specific recombinant ester hydrolyzing protein is one of the
vectors named and listed in Table 8.
The instant invention is directed to the novel nucleic acids, and the
proteins encoded for therein, isolated from the expression vectors of the
present invention. In particular, the present invention is directed
towards the nucleic acid sequence for DNA insert of said vectors, and the
the protein amino acid sequence(s) expressible therefrom.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Enzyme Characteristics. FIG. 1 depicts a sample activity profile
which characterizes and enzyme of the instant disclosure. Graph 1 depicts
the Temperature Profile of the enzyme plotting relative esterase activity
versus temperature. Graph 2 depicts the Residual Esterase Activity of the
listed enzyme plotting relative remaining activity versus time in hours,
at 25.degree. C., 40.degree. C., and 65.degree. C. Graph 3 depicts the pH
profile for the listed enzyme plotting Relative Esterase Activity versus
pH. Data for enzymes are summarized in Tables 1, 2 and 10.
FIG. 2. Kinetic analysis of E100. The enzyme displays normal Michaelis
kinetics yielding linear data with both a) Lineweaver-Burke and b)
Eadie-Hofstee analysis to give a Km=7.2.times.10.sup.-5 M and
Vmax=1.8.times.10.sup.-5 Mmin.sup.-1 using p-NP as the substrate.
FIGS. 3a-3b. Temperature and pH profiles of E100. a) Temperature profile of
E100. Plot of E100 catalyzed hydrolysis of p-nitrophenyl proprionate as a
function of temperature. Enzyme activity was determined upon exposure to
different temperatures. Initial rates of nitrophenylproprionate hydrolysis
were determined in 50 mM borate Buffer pH 8.5 equilibrated to the desired
temperature to which 0.25 mM substrate dissolved in CH.sub.3 CN was added
followed by enzyme. Rates were determined by monitoring the change in
absorbance at 405 nm and corrected for the spontaneous hydrolysis of
substrate substituting bovine serum albumin for enzyme. b) pH profile of
E100. The effect of pH on the hydrolysis of p-nitrophenyl proprionate
catalyzed by E100. The pH profile of the enzyme was determined by
preparing different buffers appropriate for the desired pH's at 10 mM
concentration. Reactions were performed by addition of the substrate (0.25
mM) dissolved in CH3CN to the buffer solution followed by the enzyme.
Reactions were incubated for 5 minutes after which the reaction was
terminated by addition of 0.1 mM PMSF dissolved in CH.sub.3 CN. The pH of
the mixture is adjusted to 8.5 by addition of 0.1 M Tris-HCl. Absorbances
are recorded at 405 nm and concentrations calculated based on the
.epsilon.=17 mM.sup.-1 cm.sup.-1 for the product nitrophenol. Formation of
products is corrected for the spontaneous hydrolysis of the substrate.
FIG. 4. The tolerance of E100 to the presence of organic cosolvents on the
hydrolysis of p-nitrophenyl proprionate as determined by relative rates.
Residual activity of the enzyme is determined in the presence of organic
solvent by measuring the initial rate of enzyme catalyzed hydrolysis of
pNP in the presence of various concentrations of CH.sub.3 CN. Reactions
are run in 50 mM Tris-HCl pH 8.5 at 37.degree. C. as described in
determination of activity. Changes in absorbance are corrected for
spontaneous hydrolysis of the substrate and the changes in extinction
coefficient of the product in the presence of organic cosolvent.
FIGS. 5A-5B. Substrates used to screen stereo- and regioselectivity.
Esterases are versatile biocatalysts in the sense that stereo- and
regio-selectivity can be mediated by substrate structure which fall into
four types. The compounds listed represent a range of different structural
features encountered in common substrates with potential importance for
the chemical intermediate industry. Several of the substrates are
commercially available in entantio- or diastereomerically pure form and
can be used in qualitative screening procedures described in the text.
Four classes of substrates most commonly associated with hydrolytic
biocatalysts for chiral centers resolution are considered. A) Type I
substrates position the desired product on the carboxylic acid side of the
product, while Type II compounds the alcohol contains the requisite
functionality. B) Type III and Type IV substrates can be considered
subsets of Types I and II, but their unique properties dictate that they
be classified separately. Type III molecules require that the enzyme
differentiates a prochiral substrate while Type IV compounds are meso
structures. These last two substrate types demonstrate the synthetic
importance of biocatalyst based resolution methods as these types of
compounds are very difficult to selectively operate upon by other chemical
means.
FIGS. 6A-6U. Nucleic acid sequence and translated protein amino acid
sequence. The isolation and cloning of the genes encoding for the enzymes
of the instant invention will result in DNA segments in which an open
reading frame (ORF) may be found which corresponds to translated protein
amino acid sequence. Alternative start codons are recognized in the art,
however the encoded protein will comprise at minimum a core protein ORF.
FIG. 6A is an isolated nucleic acid sequence, and translated amino acid
sequence which correspond to E001 (SEQ ID NO.:1 and SEQ ID NO.:2) enzyme
ORF, alternative start codons are underlined. FIG. 6B is an isolated
nucleic acid sequence, and translated amino acid sequence which correspond
to E009 (SEQ ID NO.:3 and SEQ ID NO.:4) enzyme ORF, alternative start
codons are underlined. FIG. 6C is an isolated nucleic acid sequence, and
translated amino acid sequence which correspond to E011 (SEQ ID NO.:5 and
SEQ ID NO.:6) enzyme ORF, alternative start codons are underlined. FIG. 6D
is an isolated nucleic acid sequence, and translated amino acid sequence
which correspond to E101 (SEQ ID NO.:7 and SEQ ID NO.:8) enzyme ORF,
alternative start codons are underlined. FIG. 6E is an isolated nucleic
acid sequence, and translated amino acid sequence which corresponds to
E019 (SEQ ID NO.:9 and SEQ ID NO.:10) enzyme ORF, alternative start codons
are underlined. FIG. 6F is an isolated nucleic acid sequence, and
translated amino acid sequence which corresponds to E005 (SEQ ID NO.:11
and SEQ ID NO.:12) enzyme ORF, alternative start codons are underlined.
FIG. 6G is the cloned isolated nucleic acid sequence which contains the
E004 (SEQ ID NO.:13 and SEQ ID NO.:14) ORF, alternative start codons are
underlined. FIG. 6H is the cloned isolated nucleic acid sequence which
contains the E006 (SEQ ID NO.:15 and SEQ ID NO.:16) ORF, alternative start
codons are underlined. FIG. 6I is the cloned isolated nucleic acid
sequence which contains the E008 (SEQ ID NO.:17 and SEQ ID NO.:18) ORF,
alternative start codons are underlined. FIG. 6J is the cloned isolated
nucleic acid sequence which contains the E010 (SEQ ID NO.:19 and SEQ ID
NO.:20) ORF, alternative start codons are underlined. FIG. 6K is the
cloned isolated nucleic acid sequence which contains the E013 (SEQ ID
NO.:21 and SEQ ID NO.:22) ORF, alternative start codons are underlined.
FIG. 6L is the cloned isolated nucleic acid sequence which contains the
E015 (SEQ ID NO.:23 and SEQ ID NO.:24) ORF, alternative start codons are
underlined. FIG. 6M is the cloned isolated nucleic acid sequence which
contains the E016 (SEQ ID NO.:25 and SEQ ID NO.:26) ORF, alternative start
codons are underlined. FIG. 6N is the cloned isolated nucleic acid
sequence which contains the E017 (SEQ ID NO.:27 and 28) ORF, alternative
start codons are underlined. FIG. 6O is the cloned isolated nucleic acid
sequence which contains the E020 (SEQ ID NO.:29 and SEQ ID NO.:30) ORF,
alternative start codons are underlined. FIG. 6P is the cloned isolated
nucleic acid sequence which contains the E027 (SEQ ID NO.:31 and SEQ ID
NO.:32) ORF, alternative start codons are underlined. FIGS. 6Q (SEQ ID
NO.:33), 6R (SEQ ID NO.:34), 6S (SEQ ID NO.:35), 6T (SEQ ID NO.:36) and 6U
(SEQ ID NO.:37) are partial sequences.
FIGS. 7A-G. Substrate Chain Length Specificity FIG. 7A is a graph of data
from a colorometric esterase assay performed on the substrate:
bis-p-nitrophenyl-Carbonate. FIG. 7B is data from a colorometric esterase
assay performed on the substrate: p-nitrophenyl-Acetate. FIG. 7C the
substrate: bis-p-nitrophenyl-Propionate. FIG. 7D the substrate:
bis-p-nitrophenyl-Butyrate. FIG. 7E the substrate:
bis-p-nitrophenyl-Caproate. Figure the substrate:
bis-p-nitrophenyl-Caprylate. FIG. 7G the substrate:
bis-p-nitrophenyl-Laurate. Note that E009 is an 80.times. dilution
compared to the other enzymes in b, c, d, and f.
FIGS. 8A-D. Entantiomer Substrate Specificity FIG. 8A summarizes the
results of colorometric esterase activity assays for entantiomer
specificity. FIGS. 8B-D reports quantitative colorometric assay data in
terms of minutes required for detectable color change.
FIG. 9. Enzyme Activity against para-nitroanilide compounds Table lists the
results of enzyme activity assay against various substrates. Data is
reported as normalized OD readings.
DETAILED DESCRIPTION OF THE INVENTION
The instant invention provides for isolated commercially useful protein
preparations from themostable bacteria which are selected for enzymatic
activity, and characterized by apparent molecular weight, pH, and
temperature stability. The isolated protein of the instant disclosure can
be used as molecular weight markers for finding similar enzymes, as well
as functionally as enzymes for carrying out biocatalysis. Commercial
chemical synthesis of specific racemic products often require the use of
such isolated enzyme preparations.
The results of characterization assays demonstrate that the esterase
enzymes described have a range of optimal parameters. For instance, E100
and E101 have optimal operating temperatures above 70.degree. C. as would
be consistent with enzymes isolated from an extreme thermophile, and
E001-E021 have optimal commercial temperatures in the range of
40-50.degree. C. as would be consistent with enzymes isolated from the
more moderate thermophilic organisms. Both groups, however, provide added
stability and functionality as compared to other known esterases from
thermophilic bacteria. E001-E021 provide an optimal temperature
environment for chemists who wish to work in less extreme temperature
ranges, and also function well at room temperature. The results also
demonstrate that the enzymes described posses a variety of pH optima
including some with no apparent preference under the conditions of the
experiment, however the trend for most of the proteins is to have pH
optima near or slightly below neutral.
The following examples are meant by way of illustration, and not
limitation, as to the specific embodiments of the instant invention. One
of ordinary skill in the art would understand that many equivalents to the
instant inventions can be made with no more than routine experimentation.
EXAMPLE 1
Isolation and Propagation of Thermophilic Organisms
Strains
Thermus sp. T351 (ATCC 31674) is available from the American Type Culture
Collection (ATCC). All isolated strains and cultures are grown on TT
medium (36). This medium consists of (per liter): BBL Polypeptone (8 gm),
Difco Yeast Extract (4 gm), and NaCl (2 gm). Small scale cultures for
screening are grown at 65.degree. C. at 250-300 rpm with 1 liter of medium
in a 2 liter flask. Larger scale production of cells for enzyme
purification are grown in 17 liter fermentors (LH Fermentation, Model 2000
series 1). The fermentors have a working volume of 15 liters and cultures
were grown in TT broth, 250 rpm, 0.3 to 0.5 vvm (volumes air/volume media
per minute) at 65.degree. C. Temperature is maintained by circulating
65.degree. C. water from a 28 liter 65.degree. C. water reservoir through
hollow baffles within the stirred jars. E. coli strains are grown as
described in (37).
Enrichment Procedures for Newly Isolated Thermophiles.
Multiple stream sediments, composting organic materials, and soil samples
are used to isolate new strains. These samples are collected from numerous
geographic sites ranging from the Midwest to the Southeast. Samples
(.about.1 gm) are resuspended in 2 ml of TI broth and 50-100 .mu.l of
these samples were plated onto TT agar plates containing twice the usual
amount of agar (3%). Agar is usually added to a final concentration of
1.5% for solid media This prevents highly motile microorganisms from
overcrowding the plate at the expense of other microbes. Plates are
incubated at 55.degree. C. or 65.degree. C. for one to two days and
isolates then purified by numerous restreaks onto fresh plates for single
colony isolation. The initial basis for differentiation is color, colony
morphology, microscopic examination, temperature of growth, and lipase and
esterase activities. Several hundred strains were initially isolated. 65
different microorganisms were chosen for further study.
EXAMPLE 2
Methods for Esterase Identification and Assay
Esterase Plate assay
Organisms are grown in liquid cultures on TT media at either 55.degree. C.
or 65.degree. C. Cells are pelleted by centrifugation (3,000 RPM for 20
minutes) and the supernatants saved to be tested. Pellets are washed with
2 volumes of 10 mM Tris HCl pH 8.0 three times after which the cell
pellets are resuspended in fresh Tris buffer and disrupted by sonication.
Cell debris is removed by centrifugation and the crude extracts were
tested for esterase activity on an esterase screening plate. Briefly,
fifty microliters of cell extract is transferred to a well on a microtiter
plate consisting of 0.1 mg/ml of either 5-bromo-4-chloro-3-indolyl acetate
or butyrate (for esterase activities) suspended in 0.7% agarose and 0.1M
Tris-HCl pH 8.0. Control wells consist of addition of either buffer, 20 U
of Pig Liver Esterase (PLE), or 20 U of Porcine Pancreatic Lipase (PPL).
Plates are incubated for sufficient time to allow full color development
in control wells, usually about twenty minutes at 37.degree. C. Dark wells
represent positive activity.
Both cell extracts and culture supernatants are tested for esterase
activity by this method. Only cell extracts showed significant esterase
activity.
Esterase Liquid assay and determination of specific activity
Protein concentrations are determined by the Pierce BCA assay using defined
concentrations of bovine serum albumin as the standard. Protein
concentrations are obtained from the calibrated absorbance of the sample
solutions at 562 nm and are expressed as milligrams of protein. Esterase
activities are routinely measured by determining the rate of hydrolysis of
p-nitrophenylproprionate (0.5 mM from a 10 mM stock dissolved in CH3CN) in
50 mM sodium phosphate buffer pH 7.0 equilibrated at 40.degree. C. and
monitored at 346 nm (isosbestic point for the acid/carboxylate couple
.epsilon.=4800). The specific activity is defined as the amount of
p-nitrophenol produced in micromoles per minute per milligram of total
protein.
Identification of extremely stable esterases.
Native (non denaturing) 10% polyacrylamide gels are run on crude extracts.
After electrophoresis, the gels are equilibrated in pH 7.6 Trizma buffer
and then stained for activity in either 0.15% X-acetate. The gels are then
incubated at 55.degree. C. for up to 30 minutes. These gels can then be
stained with an esterase activity stain containing either
5-bromo-4-chloro-3-indolyl acetate (X-acetate), 5-bromo-4-chloro-3-indolyl
butyrate (X-butyrate) or 5-bromo-4-chloro-3-indolyl caprylate
(X-caprylate) and produced indigo precipitates. Two major bands were
apparent in the lanes with Thermus crude extracts. A single small band of
activity is seen in the E. coli control lanes. Esterases can be identified
from Thermus sp. T351 and from several of the new isolates. Table 1
summarizes the activities which are found from these organisms.
TABLE 1
Summary of New Esterases and Strains Identified
Growth Temp (.degree. C.)
Isolation mw Specific
Isolate.sup.1 Esterase Source 37 55 65 Temp
(.degree. C.) (kD).sup.2 Activity.sup.3
S1 E001 soil nd nd + 65
22 0.011
54 E002 compost - + + 65 28
0.87
50 E003 compost - + + 65 28
2.2
GP1 E004 soil nd nd + 65
36 0.3
C-1 E005 compost nd nd + 65
28 2.3
55 E006 compost - + + 65 36
2.1
46 E007 compost - + + 65 28
0.3
30 E008 soil - + + 55 28
2.1
28 E009 soil - + + 55 36
2.0
29 E010 soil - + - 55 46.5
2.3
31 E011 soil - + - 55 36
3.6
26b E012 soil - + - 55 28
5.2
27 E013 soil - + + 55 36
2.7
34 E014 soil - + +/- 55 36
0.8
62 E015 compost - + + 55 36
3.4
47 E016 compost - + + 65 28
0.8
49 E017 soil - + + 65 36
0.03
C-3 E018 compost nd nd + 65
36 0.077
4 E019 compost - + + 55 30
0.4
7 E020 compost - + + 55 28
1.6
32 E021/17b.sup.4 soil - + +/- 55 36
0.3
Thermus sp. T351 E100 ATCC# 31674 nd + + 65 45
0.0032
Thermus sp. T351 E101 ATCC# 31674 nd + + 65 135
0.032
.sup.1 Isolates GP1, 27, 28, 29, 30, 31, 32, 34, 62 appear to be
thermophilic Actinomyces.
.sup.2 Approximate molecular weight as determined by chromatography for
E001-E021 or SDS-PAGE for E100 and E101.
.sup.3 Specific activity is the amount of p-nitrophenol produced in
micromoles per minute per milligram of total protein at 40.degree. C.
after purification to homogeneity (for E100 and E101) or semi-purification
(for E001-E021) as described in the Examples.
.sup.4 E021 is also referred to as E017b.
EXAMPLE 3
Procedure for Purification of Esterase Activity to Homogeneity
Protein Isolation
A large batch cell culture is grown according to the methods described in
Example 1 and the cell paste is collected by centrifugation and stored at
-80.degree. C. 100 g of cell paste is thawed in 200 ml of a stirred
solution composed of 50 mM phosphate buffer at pH 7.5 containing 200 mM
KCl and 0.1 mM EDTA. Once dissolved, the suspension is allowed to warm to
room temperature and then treated with lysozyme (0.1 mg/ml) for 2 hours.
The solution is then sonicated to completely disrupt the cells. Settings
used on a 375 watt Sonics & Materials Vibra Cell sonicator with a standard
1/4" horn were 5 minutes of power setting 8 disruption with a 50% pulse
rate. Alternative methods for cell disruption can include processing the
cells through a device such as a french press, Gaullen homogenizer,
microfluidizer or other homogenizer. Cell debris is removed by
centrifugation and proteins can be precipitated by NH.sub.4 SO.sub.4
fractionation to 60% saturation. Precipitated protein is centrifuged and
resuspended in minimal volume of 50 mM phosphate pH 6.5 containing 1 mM
.beta.-mercaptoethanol (BME).
DEAE Purification
The protein solution is dialyzed against the resuspension buffer 3 times
using 10 Kd pore size dialysis tubing. The resulting protein solution is
diluted two fold in the buffer and applied to a 100 ml bed volume DEAE
column equilibrated in the same buffer. The column is washed with 200 ml
equilibration buffer and then eluted with a linear gradient from 0 to 0.5
M NaCl.
Q Resin purification
Active fractions isolated from DEAE purification are pooled and dialyzed
against three changes of equilibration buffer and dialysate was applied to
a 50 ml bed volume of sepharose Q resin equilibrated with the buffer
above. The column is washed with 100 ml of 50 mM phosphate pH 6.5
containing 0.1 M KCl and 1 mM BME and then eluted with 150 ml of a KCl
gradient from 0.1 M to 0.6M added to the above buffer.
Ultrafiltration Concentration
Active fractions are pooled and concentrated using an Amicon
Ultrafiltration system fitted with a 30 Kd cut off membrane.
Preparative SDS PAGE
Concentrated protein solutions are loaded to a preparative 10% SDS-PAGE gel
using the standard SDS loading buffer without boiling the sample. After
development, the gel is treated with 0.7% agarose containing 0.1M
phosphate pH 7.5 and 0.1 mg/ml 5-bromo-4-chloro-indoylacetate. The
resulting blue band was excised from the gel, placed in dialysis tubing
and the protein is recovered by electroelution in 0.05M Tris buffer pH 8.5
for 1 hour. At this stage the protein is purified to homogeneity as
observed by both native- and SDS-PAGE stained with either coomassie or
silver stain. Protein can be stored at 4.degree. C. for future use.
Gel filtration
A gel filtration column can also be used as a further or substituted
purification step.
EXAMPLE 4
Method for Commercial Grade Preparation of Isolated Esterase
For many industrial applications, a completely purified preparation of
enzyme is neither required nor desired due to production cost
considerations. A rapid, inexpensive protocol to produce a protein of
interest in a form which is isolated to contain protein with significant
esterase activity is desired. One such semi-purification procedure is
described here. 50 g of cell paste is thawed in 100 ml of 50 mM Tris HCl
buffer at pH 7.5 containing 0.1M NaCl and 0.01 mM EDTA. Cells are
disrupted by sonication and the cell debris is removed by centrifugation.
The crude cell lysate is diluted by three fold with 50 mM Tris-HCl pH 7.5
and the material is loaded to a DEAE cellulose column (bed volume 60 ml)
equilibrated with the dilution buffer. The column is washed with three
column volumes of dilution buffer followed by a salt gradient of 0-0.5M
NaCl over 4 column volumes. Active fractions eluted from the ion exchange
resin in the salt gradient window of 0.25-0.35 M. Fractions were assayed
for activity as described under determination of specific activity and
those showing the highest activity were pooled and concentrated by
ultrafiltration with 10 Kd molecular weight cut off membrane. Concentrated
enzyme samples are stored at 4.degree. C. for further use. In some
instances, more than one ester hydrolysis activity may still be detected
under long term exposure to substrate agarose overlays of proteins
separated on native PAGE, indicating very small quantities of a second
esterase activity which should not interfere with most industrial
applications. A further purification (such as an Ammonium sulfate salt
precipitation, gel filtration, or other methods as described in Example 3)
can be applied if necessary. The process can be scaled up or down as
desired.
EXAMPLE 5
Method for Determination of Temperature Profile
Optimal temperature profiles for an esterase protein is performed by
measuring the activity of the esterase diluted into 0.1M sodium phosphate
buffer pH 7.0 equilibrated at 30.degree. C., 35.degree. C., 45.degree. C.,
55.degree. C. and 65.degree. C. respectively for five minutes. The
temperature profile is then determined by measuring the rate of hydrolysis
of p-nitrophenylproprionate added to the equilibrated solution under
reaction conditions described for determination of specific activity in
Example 2 (modified by the various temperatures used in this experiment).
Control reactions that substitute bovine serum albumin for esterase
enzymes are used to allow correction for temperature dependent
autohydrolysis of the substrate. The data is then plotted as relative
activity versus the temperature of the reaction.
EXAMPLE 6
Method for Determination of Enzyme Stability
The long term catalytic stability the esterase enzyme is evaluated by
testing the activity remaining after exposure to various temperatures. The
enzyme stock solution is diluted into 0.1 M sodium phosphate buffer pH 7.0
and placed in a temperature bath equilibrated to 25.degree. C., 40.degree.
C. or 60.degree. C. respectively under sealed conditions to avoid
concentration effects due to evaporation. Residual activity is then
determined by removing aliquots at regular intervals and measuring the
rate of hydrolysis of p-nitrophenyl-proprionate as described above.
Results are plotted as relative activity vs. time. The results indicate
that all enzymes tested retain most of the initial activity for at least
48 hours when exposed to temperatures up to and including 40.degree. C.
Activity does decrease at 60.degree. C. particularly for enzymes isolated
from organisms with optimal growth temperatures near 55.degree. C. FIG. 4
is an example of the typical data obtained. Data for enzymes are
summarized in tables 1, 2 and 10.
EXAMPLE 7
Method for Determination of pH Profile
The pH profile of an esterase is determined as follows. The rate of
p-nitrophenylproprionate hydrolysis is determined under reaction
conditions similar to those described for determination of specific
activity in Example 2 with buffers of wide useful pH windows that overlap
with at least one data point. For the purposes of these experiments two
buffers were selected that met the above criteria, Mes (useful range of
6-6.5) and Bis-tris propane (useful buffer range 6.5-9). All pH tests were
corrected for spontaneous autohydrolysis by subtraction of experimental
runs from controls substituting bovine serum albumen for esterase. This
control data treatment becomes especially important for pH's greater than
7.5.
EXAMPLE 8
Solvent Effects on Esterase Activity
Industrial applications for biocatalysts often require that enzymes
function under non-native and harsh conditions. Exposure to elevated
temperatures and pH fluctuations are possible challenges to enzyme
activity, however the lack aqueous solubility of many compounds that may
serve as substrate targets for biocatalysts is a significant challenge to
the industrial organic chemist. Organic cosolvents are commonly used in
reactions and isolated enzymes must be able to survive under conditions of
relatively high concentrations of cosolvent. Experiments are run in the
presence of various organic solvents such as ethanol, acetonitrile,
dimethylformamide, dioxane, toluene, hexane and detergents like SDS,
triton X100 and Tween 20. Additional experiments are also performed to
test the activity of isolated catalysts in nearly anhydrous solvent
conditions in which the enzymes will be lyophilized from buffers and pH's
of optimal activity.
EXAMPLE 9
Method for Protein Characterization by Migration on Native PAGE
The number of esterase enzymes in each semi-pure sample is determined from
native gel PAGE using 4-15% acrylamide gradient (precast gels purchased
from Bio-Rad laboratories) separating proteins based on their charge to
size ratio. The gel shows trace contamination with other enzymes capable
of indoylacetate hydrolysis that could not be detected easily with the
HPLC because of column dilution effects. What is clear from the gel
experiments is that most of the samples have a single major activity band
or zone that have similar migration characteristics.
EXAMPLE 10
Determination of Relative Molecular Weight by Chromatography
The estimated native molecular weights for the protein of interest is
determined by separation on a Pharmacia Superdex S200 FPLC column fitted
to a Hitachi HPLC 6200 system. Proteins were separated by isocratic
elution in 0.05 M sodium phosphate buffer at pH 7.0 containing 0.1 M NaCl.
The solvent flow rate was maintained at 0.5 ml/min and protein was
detected by UV at 280 nm. Esterase active fractions were detected
initially by 5-bromo-3-chloro-3-indolyl-acetate plate assay with follow-up
assay of most active fractions by p-nitrophenyl-proprionate hydrolysis
(both methods are described in Example 2). Molecular weights are estimated
by comparison to standard elution profiles (plotted as the log of
molecular weight vs. time in minutes) generated by use of the following
proteins: .beta.-amylase 200 Kd, alcohol dehydrogenase 150 Kd, bovine
serum albumin 66 Kd, carbonic anhydrase 29 Kd, cytochrome c 12.3 Kd.
EXAMPLE 11
Characterization of Substrate Specificities
Substrate preference of esterases for hydrolytic activity on various esters
can be determined as follows. A grid of molecules is prepared on
microtiter plates by dissolving each substrate (0.1 mM final
concentration) in CH.sub.3 CN and mixing with 0.1M phosphate buffer pH
7.5. Partially purified enzymes is then added to the wells and the
reaction mixture is incubated for 30 minutes. Crude lysates can also be
tested this way. Plates are checked after 10, 20 and 30 minutes to
determine relative activities. For experiments with noncolored substrates,
reactions are run in test tubes under the same conditions as described for
the colored substrates except that the reactions are extracted three times
with dichloromethane. The organic layers are combined, dried with
MgSO.sub.4 and concentrated to 0.1 ml in a nitrogen stream. The
concentrates are then spotted to silica gel TLC plates and developed in a
solvent mixture of 80:20:0.01 hexane:ethyl ether:acetic acid. TLC plates
are visualized with UV and I.sub.2.
EXAMPLE 12
Rapid Screen Assay for Quick Substrate Specificity Characterization
A new method was developed to rapidly screen for esterase activity based on
the mechanism of the enzyme catalyzed hydrolysis reaction wherein the pH
of the system is reduced by the release of protons upon ester hydrolysis.
The proton flux in the reaction can be monitored by use of indicator dyes
that have pH-dependent color transitions in the desired pH range of enzyme
activity. The best indicators tested are phenol red for enzymes that
function optimally at slightly elevated pHs (starting point pH 8.5) or
bromothymol blue (starting point pH 7.2) for enzymes that operate well at
more neutral conditions.
The indicator reactions are monitored by one of two methods. Spectroscopic
studies are performed by measuring the UV/Vis maxima of a 0.001% solution
of either phenol red or bromothymol blue dissolved in different pH buffers
at 5 mM concentration. Hydrolytic reactions are then performed by adding
the substrate (0.1 mM final concentration) to a 5 mM buffer solution
(sodium phosphate pH 7.2 for bromothymol blue indicator and sodium borate
pH 8.5 for phenol red indicator) and equilibrating the temperature at
25.degree. C. for five minutes followed by initiation of the reaction by
addition of 0.1 U target enzyme.
An alternative method for monitoring the hydrolytic reactions is useful for
broad screening applications. 5 mM buffer containing 0.001% indicator dye
and substrates dissolved in CH.sub.3 CN, DMF or DMSO to an organic solvent
composition of no more than 10% is added to a stirred 24 well microtiter
tray. The temperature is allowed to equilibrate for five minutes at
25.degree. C. after which the reaction is initiated by addition of 0.1 U
of the esterase. Reaction progress is monitored by solution color changes
upon which, aliquots of NaOH are added to return the reaction color to the
starting point. Reactions are determined to be complete when no further
color change is detected after prolonged incubation. Product formation is
verified by TLC analysis of reactions acidified with 0.1 M HCl, extracted
with ethyl acetate, dried with Na.sub.2 SO.sub.4 and concentrated under a
stream of N.sub.2. For testing substrates in which enzyme-based chiral
resolution is being screened, products are separated and isolated by
chiral phase HPLC and enantiomeric purity is determined by integration of
peak areas for each isomer.
Rapid assay of a variety of hydrolytic activities, in this cases esterases,
is determined in a microtiter plate experiment using several different
enzymes and substrates. Accurate comparison of commercially available
enzymes can be insured by using the same specific activity for each enzyme
determined from the total protein and the initial rate of hydrolysis of
the common substrate p-nitrophenylproprionate. The data are recorded as
the time required to visualize a pH dependent color change for the given
indicator dye. Control experiments using BSA as the protein source cause
no change in indicator color and establish that pH changes in solution are
the result of an enzyme catalyzed hydrolysis. Control tests of reaction
solutions containing enzymes and indicators without substrates established
that color changes in the solutions are not the result of buffer salts or
the enzymes alone.
Studies performed to determine whether the microtiter plate format was
amenable to small scale preparative chemistry are performed as follows.
Using racemic phenethylacetate and pig liver esterase, reactions are run
and titrated with aliquots of 0.1N NaOH to maintain original solution
color until no further color changes occurred at which point the reactions
are stopped. Products are isolated and tested by TLC and compared to total
amount of base added to verify the extent of the reaction. Phenethyl
alcohol is separated from starting acetyl ester by flash column
chromatography followed by analysis by chiral phase HPLC. The enantiomeric
excess of the hydrolysis products is determined from the peak integration
and compared to an identical reaction run in the absence of indicator dye.
The results from these experiments suggest that inclusion of indicator dye
has no effect on the stereoselectivity of esterase catalyzed resolution of
phenethylacetate.
In order to test the assay for usefulness in a broad-based enzyme screening
method, seven organisms isolated from various sources in the environment
were tested for their ability to produce enzymes that would catalyze the
hydrolysis of a group of structurally diverse compounds. Table 2 shows the
results of these studies.
TABLE 2
Substrate Specificity.
Lysate Hydrolytic Rate (min)
Substrate N/E E001 E003 E004 E005 E006 E016 E017
E018
##STR2##
-- 60 240 20 <5 <5 -- -- 15
##STR3##
-- 60 20 <5 <5 <5 <5 120 60
##STR4##
-- -- 240 -- 120 240 -- 300 --
##STR5##
-- -- 300 240 240 240 -- -- 240
##STR6##
-- 240 240 20 60 60 120 900 60
Solvent Control -- -- -- -- -- -- -- -- --
Results are reported as the amount of time required to change indicator
color. The data is indicative of variable substrate specificity between
different environmental isolates. Of particular note is the suggestion of
stereoselectivity as determined from the relative rates of hydrolysis for
substrate enantiomers. Control reactions are similar to those described
above in the substrate specificity studies with commercially available
enzymes.
EXAMPLE 13
Further Characterization of Substrate Specificities
Depicted in FIG. 10 are examples of the substrates that can be tested with
each enzyme activity. These molecules have been chosen specifically
because of their importance as intermediates in the synthetic literature
with the potential for industrial application. Experiments can be
performed with crude lysates or proteins isolated from media broth in
cases where the activities are known to rapidly assess the likely reaction
chemistry including substrate preference and stereochemistry. All
structure activity tests are compared to standard mesophile biocatalysts
such as pig liver esterase. The reactions are monitored by TLC analysis to
compare the products to standards purchased from commercial sources or
prepared by chemical means (for example, base-catalyzed hydrolysis of
esters).
Investigations of stereochemical preference by each esterase can be
evaluated by one of two methods. In the first method, standard single
stereoisomers of commercially available entantiomerically pure substrate
esters are hydrolyzed by each enzyme and the relative rates of hydrolysis
for each antipode are used as diagnostic qualitative determinants of
potential chiral selectivity. In the second method, those molecules not
commercially available as single stereoisomers are hydrolyzed as racemates
using kinetic resolution methods (running the reaction generally less than
50% completion). The products of the reaction are isolated and analyzed
for their enantiomeric excess (ee) by chiral phase HPLC (Diacel Chiralcel
OD or OB) or .sup.1 H NMR of the corresponding diasteriomers prepared by
derivatizing products to Mosher derivatives (alcohol products) or menthyl
derivatives (carboxylate products). Diastereomeric ratios determined from
the NMR spectra are based on corresponding peak integrations and compared
to either literature values or standards obtained from commercial sources
using of chiral shift reagents when necessary. Optical rotations and
absolute configurations of the products are then determined by
polarimetric analysis and compared to values found in the literature or
determined from standards obtained from commercial suppliers.
EXAMPLE 14
Characterization of Proteins E001-E021/17b
Strains from the identified sources as listed in Table 1 were isolated by
growth in TT media at 65.degree. C. as described in Example 1 (ie. S1 from
soil, etc.). Specific esterase hydrolytic activity was identified by the
methods described in Example 2 and the isolated esterase protein assigned
the identifier as listed in Table 1 (ie. E001 etc.) To prepare enzyme, a
15 liter culture of isolate is grown and the cells are spun down and
collected as described in Example 1. The cells are lysed and a isolated
preparation of was purified according to the procedures outlined in
Example 4. The protein was characterized using the methods described in
Example 5 to determine the temperature profile, Example 6 to determine
protein stability, and Example 7 to determine the pH profile, and the
results are shown in FIG. 4. The protein was characterized by migration on
Native gradient PAGE as described in Example 9 and the data is shown in
FIG. 2. The specific activity was determined as described in Example 2 and
the molecular weight was determined by chromatography as described in
Example 10 and are presented in Table 1. Substrate specificity for several
proteins has been demonstrated and are shown in Table 2. Thus the
identified and characterized esterases have been demonstrated to be
useful, and to posesses unique activity at commercially useful purity.
Certain results are summarized in Table 10.
EXAMPLE 15
Characterization of E100
Purification of E100
E100 is purified from Thermus sp. T351 over 300 fold by a series of four
steps described in Example 3: DEAE purification, Q Resin purification,
Ultrafiltration concentration, and preparative SDS PAGE. The specific
activity could not be measured in the crude lysate since there was a
secondary esterase activity present (E101). The secondary activity could
be completely removed from the target esterase during the first
chromatographic step in which the secondary esterase passed through the
DEAE column unbound. For purification of various technical grades of E100,
DEAE purification alone is sufficient to yield E100 enzyme substantially
purified away from any other contaminating activity. Q Resin purification
and ultrafiltration allow for higher purity product to be produced as
required by specific applications. A final SDS PAGE purification step
allows the protein to be purified to homogeneity for determination of
molecular characteristics.
Protein Characterization
The active band is collected by electroelution on a preparative SDS-PAGE
gel and rerun on 10% SDS-PAGE under denaturing conditions. This shows a
single band with a relative molecular mass of about .about.45 Kd. Unboiled
samples run on the same SDS-PAGE gels show multiple bands in approximate
increments of the proposed monomeric molecular mass. Additionally, the
nonboiled sample can be stained for activity, however only bands
corresponding to multimeric forms of the enzyme are found to retain
activity beginning with dimeric species. The specific activity of the
purified protein is approximately 3.2.times.10.sup.-6 Mmin.sup.-1
mg.sup.-1 using 4-methyl-umbelliferyl-butyrate (MUB) as the substrate.
Measurement of E100 Enzyme Activity
Esterase activity is measured by monitoring the hydrolysis of
p-nitrophenylproprionate (pNP), or in some cases MUB. Each substrate is
dissolved in acetonitrile and added to the reaction mixture (100 .mu.M
final concentration) which contain 50 mM Tris HCl pH 8.5 adjusted for
temperature dependent pH variation. Reactions are thermally equilibrated
at 37.degree. C. for 5 minutes prior to initiation of the reaction by
addition of 10 .mu.L of enzyme sample, while control reactions substituted
equivalent amounts of BSA. The reaction is monitored
spectrophotometrically at 405 nm .epsilon.=17 mM.sup.-1 cm.sup.-1 for pNP
and 360 nm .epsilon.=7.9 mM.sup.-1 cm.sup.-1 for MUB.
The rates of enzyme catalyzed hydrolysis are corrected for the spontaneous
hydrolysis of the substrate. Protein concentrations are determined by
either the absorbance at 280 nm or by Lowery assay. Crude activity is
determined by a calorimetric assay based on the hydrolysis of
5-bromo-4-chloro-3-indoyl esters suspended in a 0.7% agar matrix on
microtiter plates. A 0.1 mg/ml solution of the indolyl derivative is
dissolved in a minimal volume of acetonitrile and added to a warm solution
of 0.7% agar containing 0.1M phosphate buffer pH 7.5. 10 .mu.L of this
solution is distributed to microtiter plates which, when cooled, could be
used with as much as 100 .mu.L of enzyme sample and incubated at
temperatures from ambient to >65.degree. C.
E100 was effectively inhibited when exposed to tosyl fluoride but was
unaffected by the presence of either metal ions, chelating agents or
reducing molecules Table 3.
TABLE 3
Inhibition by reaction components on the hydrolysis of p-
nitrophenylprorionate by E100
Additive (concentration) Relative Rate.sup.a (%)
None 100
PMSF (0.1 mM) 0
BME (10 mM) 99
DTT (1 mM) 101
CaCl.sub.2 (10 mM) 108
MgCl.sub.2 (10 mM) 95
ZnCl.sub.2 (10 mM) 90
EDTA (1 mM) 96
Reaction conditions are those described in the general experimental above
except for the addition of specified components. Relative rates are
corrected for the spontaneous rate of hydrolysis of the uncatalyzed
reaction.
Substrate specificity of E100
The substrate specificity was tested as outlined as according to Example
11, and the results from the structure activity experiments for E100 are
shown in summary Table 4. E100 displays a broad substrate specificity
catalyzing the hydrolysis of a number of nitrophenyl, coumaryl and alkyl
esters. The enzyme displays hydrolytic activity towards both straight
chain and aromatic moieties on the carboxylate side of substrates however,
carboxylate R groups of long alkyl chains >C8 or those containing naphthyl
leaving groups are not substrates. The enzyme displays no significant
activity towards either casein or milk as assayed by clearing zones on
agar plates.
TABLE 4
Substrate Activity of E100
Substrate E100 Control
I-acetate.sup.a ++ -
I-butyrate.sup.a ++ --
I-caprylate.sup.a + --
N-acetate.sup.a -- --
U-acetate.sup.a ++ +/-
U-stearate.sup.a -- --
pN-acetate.sup.a ++ --
pN-proprionate.sup.a ++ --
oN-proprionate.sup.a ++ --
oN-caprylate.sup.a + -
oN-palmitate.sup.a +- -
oN-stearate.sup.a - --
Me-PA.sup.b + --
Et-PA.sup.b + --
isoProp-PA.sup.b + --
Structure activity assay of partially purified esterase E100 from Thermus
species. (++) highest activity as determined by .sup.a color formation in
less then 10 min or significant product formation on .sup.b TLC. The
remaining activity measurements follow the order: + > +/- > - > --.
Structure abbreviations are as follows: I, chloro-bromo-indoyl, N,
a-napthyl, U, methylumbelliferyl, pN, p-nitrophenyl, oN, o-nitrophenyl,
PA, phenylacetate.
Determination of Kinetic Characteristics
Kinetic characteristics are determined by measuring the concentration
dependent initial rates of enzyme catalyzed hydrolysis of nitrophenyl
proprionate. Reactions are run at pH 8.5 in 50 mM Tris-HCl buffer
equilibrated to 37.degree. C. and initiated by addition of enzyme. Rates
are determined from the absorbance changes due to formation of product
nitrophenol at 405 nm. Rates are corrected for the spontaneous hydrolysis
of substrate during the course of the reaction. Concentration vs. rate
data are analyzed by both double reciprocal plots and by HanesWolff plots
to determine Km, Vmax and Vmax/Km. The kinetic characteristics of E100
determined from plots of the initial rates of hydrolytic reactions are
shown in FIG. 6.
Determination of Temperature Profile and Optimal pH for E100
The temperature profile of the enzyme is determined as shown in FIG. 7a.
Enzyme activity is observed to steadily increase to the limit of the
assay, over 70.degree. C., (where the background signal from
autohydrolysis of the substrate became too high and is no longer
correctable) as the temperature of the reaction is elevated and suggests
that the low end for optimal activity for E100 is greater than 70.degree.
C. E100 displays a basic pH profile with a low end optimal activity
observed to be approximately 9.0, the limit of substrate stability at
37.degree. C. (FIG. 7b).
Determination of Enzyme Stability in the Presence of Organic Solvents
E100 is tested for tolerance to organic solvent composition using the polar
aprotic cosolvent acetonitrile as a preliminary system. the enzyme
retained 50% of its activity in a solvent mixture of 20 vol % organic
cosolvent (FIG. 8).
N-Terminal Sequencing of E100
Purified proteins are run on 10% SDS-PAGE gels and then transferred to PVDF
membranes by electroblotting. Membranes are washed with several changes of
doubly distilled water to remove any remaining SDS or other contaminants
and then stained with coomassie blue. Membranes were then destained with
several changes of 50:40:10 MeOH:H.sub.2 O:AcOH followed by one wash of
10% MeOH. Membranes are then air dried and then submitted for sequencing.
The N-terminal sequence of E100 was determined at the University of
Illinois Urbana Champaign genetic engineering facility.
The N-terminus of E100 was determined by automated sequencing of the
polypeptide purified by 10% SDS-PAGE and transferred to a PVDF support.
The sequence obtained was: MKLLEWLK?EV, where the letters refer to the
standard amino acid single letter code and the "?" refers to an
indeterminate amino acid. Thus, E100 has been demonstrated to be a useful
esterase with unique activity at commercially useful purity.
EXAMPLE 16
Characterization of E101
E101 is one of two esterase activities that are isolated from Thermus sp
T351. E101 can be purified away from a second esterase, E100, in an early
purification step.
Purification of E101
A Thermus sp. T351 supernatant prepared as described in Examples 1 and 2 is
fractionated with NH.sub.4 SO.sub.4 and the precipitated proteins are
collected between 20-60% saturation. Pellets are redissolved in 30 ml of
buffer (50 mM Tris-HCl pH 8.0, 1 mM BME) and dialyzed against the same
buffer using 30 Kd cutoff dialysis tubing. Dialysate is loaded to 100 ml
bed volume of DEAE resin equilibrated with the buffer above and the column
was washed with 150 ml of the equilibration buffer. Active protein is
observed in the load and wash fractions, pooled, and concentrated with the
use of an Amicon concentrator fitted with a YM30 membrane. Concentrated
proteins are then loaded directly to a 25 ml bed volume of sepharose SP
resin equilibrated with the above buffer. Active fractions appear in the
load and wash fractions which are pooled and concentrated as above.
Concentrate is then loaded to a Sephracryl HR200 gel filtration column
(1.times.40 cm) and 0.5 ml fractions are collected at a flow rate of 2
ml/hr. Active fractions are collected and analyzed by SDS-PAGE. In order
to perform N-terminal sequencing, fractions considered to be homogeneous
are concentrated and submitted to a protein sequencing service center. The
enzyme is stored at 4.degree. C. for future use.
E101 can be purified over 35 fold by these methods and possesses
characteristics dramatically different from E100, the other esterase which
is isolated from this strain. Attempts to use ion exchange chromatography
result in subtractive purification since in no instance was the protein
retained. Resins investigated include DEAE, Q sepharose, CM cellulose, SP
sepharose and hydroxyappatite under conditions that varied from pH 6.0 to
9.0, and buffers from phosphate to borate including Tris and Hepes. After
two ion exchange steps the protein is purified to homogeneity by gel
filtration chromatography however, the protein appears to have an
interaction with the column as retention is considerably longer than the
molecular weight would suggest. The molecular weight of the protein
appears to be approximately 135 Kd with a monomer mass of .about.35 Kd as
determined from native and denaturing SDS-PAGE respectively.
E101 Characteristics
The specific activity of the enzyme is ten fold greater than observed for
E100 with 4-methyl-umbelliferyl butyrate (MUB) as the substrate. E101 is
inhibited by PMSF but is insensitive to metal ions or metal ion chelators.
The specific activity of the purified protein was found to be
3.2.times.10.sup.-5 mol min.sup.-1 mg.sup.-1 and was determined from
initial rates of hydrolysis using methyl umbelliferyl butyrate as a
substrate. Table 5 outlines the inhibitory effect of various substances on
E101 activity.
TABLE 5
The inhibitory effect of reaction components on the hydrolysis of
p-nitrophenylprorionate by E101
Additive (concentration) Relative Rate.sup.a
None 100%
PMSF (0.1 mM) 0
BME (10 mM) 96
DTT (1 mM) 98
CaCl.sub.2 (10 mM) 102
MgCl.sub.2 (10 mM) 97
ZnCl.sub.2 (10 mM) 100
EDTA (1 mM) 93
Reaction conditions are those described in the general experimental above
except for the addition of specified components. Relative rates are
corrected for the spontaneous rate of hydrolysis of the uncatalyzed
reaction.
Substrate specificity of E101
The substrate specificity of E101 was determined as described in Example
11. The results from the structure activity experiments for E101 are shown
in Table 6. The hydrolytic activity of the enzyme is similar to that
observed for E100 and has no observable protease activity toward milk or
casein.
TABLE 6
Substrate Activity of E101
Substrate E101 Control
I-acetate.sup.a ++ -
I-butyrate.sup.a ++ --
I-caprylate.sup.a + --
N-acetate.sup.a -- --
U-acetate.sup.a ++ +/-
U-stearate.sup.a +/- --
pN-acetate.sup.a + --
pN-proprionate.sup.a + --
oN-proprionate.sup.a ++ --
oN-caprylate.sup.a +/- -
oN-palmitate.sup.a +/- -
oN-stearate.sup.a - --
Me-PA.sup.b ++ --
Et-PA.sup.b ++ --
isoProp-PA.sup.b + --
Structure activity assay of partially purified esterase E101 from Thermus
species. (++) highest activity as determined by .sup.a color formation in
less then 10 min or significant product formation on .sup.b TLC. The
remaining activity measurements follow the order: + > +/- > - > --.
Structure abbreviations are as follows: I, chloro-bromo-indoyl, N,
a-napthyl, U, methylunmbelliferyl, pN, p-nitrophenyl, oN, o-nitrophenyl,
PA, phenylacetate.
Thus, E101 has been demonstrated to be a useful esterase with unique
activity at commercially useful purity.
EXAMPLE 17
Cloning of Esterase
General Cloning Strategy
The .lambda. ZAP cloning system from Stratagene.TM. can be used for the
library constructions and detection of esterase activity. Other cloning
systems can also be used to yield similar results. The usual efficiency of
cloning in .lambda. vectors vary from 10.sup.5 to 10.sup.7 hybrid clones
per mg of cloned DNA and is sufficient to produce a representative gene
library from a convenient amount of size-selected chromosomal DNA
fragments. We have found that detection of esterase activity in phage
plaques, as opposed to bacterial colonies, is more efficient due to the
easier access of substrate to the enzyme. Phages are generally less
sensitive to the toxic action of cloned proteins and are also able to
survive at the temperatures up to 70.degree. C. The ability of the cloning
system to tolerate elevated temperatures and potential toxicity of the
cloned proteins is necessary for the detection of the activity of
thermophilic proteins, such as the esterases described here.
Isolation of DNA for Construction of gene banks
Genomic DNA is prepared from a culture of the appropriate strain containing
the esterase of interest as described in Example 1. Cells of different
strains are grown to late log phase in 100 ml TT broth (8 g Polypeptone
(BBL 11910), 4 g yeast extract, 2 g NaCl, per liter) at 55.degree. C. or
65.degree. C. overnight shaking at 250 RPM. Cells are recovered by
centrifugation and the pellet is resuspended in 5 ml of lysis buffer (10
mM Tris-HCL, pH 7.0, 1 mM EDTA, and 10 mM NaCl). Lysozyme is added to a
final concentration of 2 mg/ml. Cells are incubated at 37.degree. C. for
15 minutes followed by the addition of SDS to 1%. The lysate is gently
extracted three times with phenol/chloroform/iso-amyl alcohol (25/24/1)
and the DNA spooled from a 95% ethanol overlay of the aqueous phase.
One of ordinary skill would find other methods for preparation of DNA which
are well known in the art (37). For example, fresh colonies of a strain
containing the esterase of interest are inoculated in 50 ml of TT media in
250 ml Erlenmeyer flask and incubated at 55.degree. C. for 24 hours at 200
rpm in a New Brunswick Environmental Shaker. The cells are harvested by
centrifugation at 3000 g for 15 min., resuspended in 5 ml of GTE buffer
(50 mM Glucose, 25 mM Tris-HCl pH 8, 10 mM EDTA) and treated with 2 mg/ml
of lysozyme at 37.degree. C. for 10 min. Lysozyme-generated spheroplasts
are lysed by the addition of 1% SDS and partially deproteinased by
addition of 100 .mu.g/ml of proteinase K at 24.degree. C. for 10 min.
Chromosomal DNA is further purified by three phenol/chloroform
extractions, precipitated with 2.5 volumes of ethanol and resuspended in 1
ml of TE (10 mM Tris pH 8.0; 1 mM EDTA), after washing in 20 ml of 75%
ethanol. The extracted fraction consists of DNA fragments larger than 50
kb, with a concentration of about 0.5 ng/.mu.l, as detected by gel
electrophoresis using a 0.7% agarose gel run at 10 V/cm for 4 hours.
Construction of Gene Libraries
Genomic DNA is partially digested with the restriction enzyme Sau3A and
then ligated to predigested Lambda ZAP Express (Stratagene Cloning
Systems). Products of ligation reactions are packed in vitro using
.lambda. packaging extracts which are purchased from Promega. This vector
accommodates DNA up to 12 kb in length and allows identification of clones
both by expression off the T3 and T7 promoters and by probe hybridization
to plaques. The library is retained and screened for esterase activity.
Other methods for generating genomic DNA libraries are also well known in
the art.
Five samples of 10 .mu.g of chromosomal DNA of each of the strains prepared
as described above, are treated with different concentrations of Sau3A
restriction endonuclease (New England BioLabs) according to the
manufacturer's instructions for 30 min at 37.degree. C. in a volume of 50
.mu.l each. The concentration of Sau3A is varied from 0.1 u to 0.002
u/.mu.g of the digested DNA in separate tubes. The reactions are stopped
by heat inactivation of the endonuclease at 70.degree. C. for 10 minutes
and analyzed by gel electrophoresis on a 0.7% agarose gel run at 10 V/cm
for 4 hours (a typical digestion pattern is obtained, data not shown).
Fractions with an average fragment size of 5 kb are chosen for cloning.
For native strains containing E001, E002, E003, E006, E007, E008, E009,
E010, E012, E016, E020 these are the second of the five samples of
digested chromosomal DNA with the concentration of Sau3A of about 0.02
u/.mu.g of the DNA. For the rest of the strains, the proper degree of
partial digestion is achieved in the first test tube with 0.1 u of
Sau3A/.mu.g of the DNA. Fifty ng of chromosomal DNA fragments are ligated
with equimolar amounts of dephosphorilatyed BamHI-arms of the lambda ZAP
phage vector (Stratagene) in 5 .mu.l with 1 unit of ligase (New England
Biolabs). Ligation reactions are performed at 1 8.degree. C. for 8 hours
and stopped by heat inactivation at 70.degree. C. for 10 min. One .mu.l of
the ligation reaction, containing approximately 10 ng of DNA insert, is
used for in vitro packaging with 10 .mu.l of lambda proheads (produced by
Promega Corp). The packaging reaction is performed at 28.degree. C. for 90
min, combined with 100 .mu.l of an overnight culture of E. coli XL1 Blue
and plated using 2 ml of 0.7% top agar (0.8% NaCl, 10 mM MgSO4) per plate
onto five 90-mm Petri plates containing LB media Serial dilutions of the
packaging mixture are produced in order to determine the cloning
efficiency which is generally about 1.0.times.10.sup.7 hybrid phages/.mu.g
of cloned DNA. Cloning efficiencies for each individual strain varied, the
size of the library generated fell within a range of 0.5 to
2.5.times.10.sup.5 from which two to twelve positive clones were analyzed
(data not shown). Hybrid phages from one plate are harvested to collect
the amplified library, which is stored in 3 ml of LB media with 25%
glycerol. The four other primary plates are treated with indicator agar
containing 5-bromo-4-chloro-3-indolyl-acetate (X-Acetate) as described
below, to find hybrid plaques carrying esterase genes.
Screening of gene banks for esterase activity
The products of the above packaging reactions are infected into E. coli XL1
blue MRF' (Stratagene). Primary plaques of an unamplified gene library are
screened for enzyme activity by overlaying the plates with top agar
containing X-Acetate for 30 minutes at 65.degree. C. The concentration of
substrate in the indicator overlay is diluted from a 4% stock in ethanol
or N,N-dimethyl formamide to a concentration generally between 0.1 and 1%
(usually about 0.4% is used) in the final solution. Other suitable
substrates may be substituted in this procedure including, but not limited
to, 5-bromo-4-chloro-3-indolyl-butyrate (X-butyrate),
5-bromo-4-chloro-3-indolyl-proprionate (X-proprionate),
5-bromo-4-chloro-3-indolyl-stearate (X-stearate),
4-methylumbelliferyl-acetate (MUA), 4-methylumbelliferyl-butyrate (MUB),
4-methylumbelliferyl-proprionate (MUP), or other
5-bromo-4-chloro-3-indolyl- or 4-methylumbelliferyl-esters which may be
either synthesized or purchased from a commercial vendor such as Sigma
Chemical. In order to inactivate background endogenous esterase activity
from E. coli, the plates are preheated at 65.degree. C. for 20 minutes.
Hybrid phages surviving this procedure are picked and re-screened three
times. The extracts are then analyzed for the presence of a protein band
with the same mobility as the native protein as described below. The
lambda ZAP cloning system permits an excision of smaller plasmid vector to
simplify the insert characterization. While other methods may be employed
for screening gene banks for esterase activity, i.e. isolation,
purification, and N-terminal sequencing of protein; creation of degenerate
nucleotide probes from N-terminal sequence; screening of gene bank with
degenerate probes, the instant method is efficient and uniquely suited for
the purpose of isolation of promising clones.
In particular, the four primary plates with phage colonies generated during
the cloning described above, are incubated at 65.degree. C. for 30 min. in
order to inactivate some of the potential E. coli esterase activities.
Approximately two ml of 0.7% top agar (0.8% NaCl, 10 mM MgSO.sub.4)
containing about 1 mg/ml of the colorimetric esterase substrate X-Acetate
or other substrate (including but not limited to X-butyrate,
X-proprionate, X-stearate, and 4-methyl-umbelliferyl based substrates) is
overlaid onto each plate. Expression of cloned esterases can be detected
by blue halos around phage colonies (or fluorescent halos in the case of
the 4-methylumbelliferyl substates). A typical result for this process can
yield a ratio of 1:3000 positive colonies to hybrid phages.
Between two and twelve primary positive phage plaques are generally picked
up from each set of plates, resuspended in 50 .mu.l of LB medium, and
streaked onto a lawn of E. coli XL1 Blue using sterile paper strips. These
purified phage plaques are then overlaid by indicator agar containing
X-Acetate as before, and positive plaques were selected as in primary
screening experiment. Three rounds of such purification by restreaking are
generally sufficient to produce a pure hybrid phage clone expressing
esterase activity. All these clone candidates demonstrate significant
esterase activity in the X-Acetate plate assay. Several clone candidates
from each strain are chosen for further analysis, each representing the
progeny of single primary phage plaque.
Testing Protein Profiles Produced by Phage Clones
Production and analysis of protein from the phage clones is performed as
follows, but alternative methods are possible: A single plaque from each
clone is resuspended in 20 .mu.l of an overnight culture of E. coli XL1
Blue (grown in LB medium with the presence of 10 mM of MgSO.sub.4),
incubated for 20 min at 24.degree. C. in one well of a 96-well microtiter
plate to allow adsorption, transferred into 15-ml test tube containing 2
ml of LB, and grown overnight at 37.degree. C. in a New Brunswick
Environmental Shaking incubator set at approximately 300 rpm. Cell debris
can be removed by centrifugation at 12,000 g for 10 min. Phage lysates
from the clones are then subjected to 4-15% gradient Native polyacrylamide
gel electrophoresis (PAGE) for comparison to the native proteins purified
from the original organisms. Precast gradient gels are purchased from
BioRad Laboratories (catalog number 161-0902) and used according to the
manufacturer's instructions for native gels. An esterase preparation from
the original strain, purified by HPLC to a single protein band is used as
a control on the same gel. Alternatively, a native protein preparation
which has not been purified to homogeneity but is purified to a single
esterase activity can be used as a control. Protein bands possessing an
esterase activity can be detected by applying an X-Acetate overlay and
incubating at room temperature for 5-20 min. The relative mobility of the
clone candidates can be compared to the native esterase protein.
The data generated for 107 hybrid phage clone candidates from 20 strains
are summarized in Table 7, which shows the results of the typical
comparison of the esterase activities detected in lambda clones compared
to the host strain. For each gene library screened, there is at least one
clone candidate expressing an esterase protein with the mobility of the
protein purified from the original strain. Several of the .lambda. clone
candidates express esterase activities which have mobilities that are
different from the major component of the esterase specimens purified from
the original strains. Similar sized bands possessing esterase activity are
observed in the native organism as minor components (data not shown).
These cloned ester hydrolyzing activities are given names depicted in
Table 7.
Excision of the Plasmid Vector from the Phage
The lambda ZAP vector allows the phage clone to be conveniently converted
into a plasmid vector to allow better physical characterization of the DNA
insert and regulated expression of cloned genes. Induction of M13-specific
replication by co-infection with the helper phage results in excision of a
multi-copy plasmid carrying the cloned insert. 10 .mu.l phage stocks of
the lambda hybrids (with about 10.sup.7 Colony Forming Units (CFU)) and 1
.mu.l of Exassist M13 helper phage (about 10.sup.10 CFU) are used to
infect 20 .mu.l of an overnight culture of the E. coli XL1 Blue grown in
LB. After 20 min at 24.degree. C., the cell suspension is transferred from
one of the wells of a 96-well microtiter plate into a 15-ml culture tube,
diluted with 2 ml of LB, grown overnight at 37.degree. C. and 300 rpm,
heated at 65.degree. C. for 10 min, and cleared by centrifugation at 3000
g for 20 min. Excised plasmids packed in M13 particles are transduced into
a lambda resistant strain, XLOLR, that does not permit the development of
the M13 helper phage. Ten .mu.l of excised phage lysate are mixed with 30
.mu.l of the overnight culture of the E. coli XLOLR strain in one well of
96-well microtiter plate, incubated for 20 min at 37.degree. C. to allow
adsorption, diluted with 100 .mu.l of LB, and incubated at 37.degree. C.
for 40 min to express the kanamycin (Km) resistance marker (neo) of the
plasmid. Cells are plated onto two LB plates supplemented with 40 mg/ml
Km. One of the plates also contains 50 .mu.l of a 4% X-Acetate stock
solution.
Preliminary experiments are performed by growing plates at 37.degree. C. to
demonstrate that a significant phenotypic segregation occurs with the
transductant E. coli colonies expressing cloned thermophilic esterases. In
an extreme case of the CE020 strain, very few colonies not expressing any
esterase activity could be re-streaked from primary transductant colonies,
which actively expressed esterase activity. Because of this segregation
and apparent instability of plasmids containing the active clones,
protocols for manipulation of most of the esterase clones needed to be
modified as compared with the standard protocol of plasmid excision
recommended by Stratagene. It was possible that the instability was due to
the function of the cloned protein expressed in the cell, thus it was
hypothesized that lowering the growth temperature might overcome the
segregation problem, since the esterases were from thermophilic organisms
and may not be as active at the lower temperatures.
Therefore, to overcome the problem of instability due to the activity of
the esterase containing plasmids, cultivation of E. coli cells harboring
thermophilic esterases is performed at 28.degree. C. and 30.degree. C.,
with the result that the effective phenotypic segregation is reduced.
Thus, in the event that a cloned thermophilic esterase activity is lethal
or partially lethal to the host cell, the growth temperature of the strain
should be lowered to 30.degree. C. or even room temperature. The
recombinant strains harboring plasmids with active esterase proteins often
exhibited a phenotypic segregation of the esterase activity on X-acetate
plates. This segregation could be due to plasmid or insert loss if the
esterase activity had toxic properties to the cell. To overcome this cells
could be grown at lower temperatures (presumably reducing the activity of
the cloned thermophilic esterases). Thus strains can be plated with
X-Acetate at 28.degree. C. and 37.degree. C. Yellow colonies of faster
growing segregants are visible at both temperatures, but contra-selection
at 37.degree. C. is much stronger. After determining that temperature
makes a large difference in stability of the clone phenotype, further
experiments are carried out by plating all plasmid based clones at
26.degree. C., generally for 48 hours. E. coli cells are plated in a
medium containing X-Acetate to detect expression of cloned esterase by the
plasmid, and a degree of segregation in or between primary colonies. Thus,
growth of the transformed cells at a temperature which reduces the
activity of the cloned esterase is important to the effective isolation of
productive plasmids.
In the specific case, eight bacterial colonies derived from each of the
phage clones are picked from the plates without X-Acetate, transferred
into 100 ml of LB supplemented with 40 mg/ml Km in a 96-well plate and
grown overnight. Progeny of these colonies are analyzed by a spot-test
using X-Acetate containing agar. Several plasmid clones derived from each
phage are chosen for further study by picking ones producing brightest
blue halos and least amount of the esterase.sup.- segregants.
Selection for the Stable Plasmid Variants
Since it is determined that the plasmid-based vectors carrying esterase
genes are often unstable, stable variants of the plasmids are isolated.
One method for such isolation is as follows E. coli cells carrying excised
plasmids are purified using LB plates supplemented with Km and a limited
amount of X-Acetate to reduce any potential negative growth impacts from
production of the somewhat lethal indole product of the calorimetric
reaction. Colonies are selected by their phenotype (in general giving a
modest growth rate and intensive blue color) and grown in 2 ml of LB with
Km in 15 ml test tube for 48 hours to reach OD.sub.600 of about 1.0 and
harvested by centrifugation at 12,000 g for 1 min. Cell pellets are
resuspended in 500 ml of 0.1 M Phosphate buffer pH 7.0 and sonicated using
a Sonics & Materials Vibra Cell 375 Watt sonicator at 4.degree. C.
Sonication is performed using a microtip, 40% max capacity, 50% time pulse
for 45 sec. Lysates are centrifuged at 12,000 g for 5 min and tested for
its relative esterase activity. Variants with the highest activity are
selected for the next round of growth and analysis. Three rounds of
plating followed by growth in liquid medium and activity assays are
performed to verify the stability of the clones.
Deviations in specific esterase activity among variants from the same
plasmid lineage can be reduced to a factor of three from over a factor of
100 by this procedure. Stabilization of the activity generally occurs at
the level corresponding to the highest activity values detected in the
first round of stabilization. This could indicate that E. coli host
mutations are being selected which allow higher tolerance of the cloned
protein, rather than simply suppressed activity of cloned toxic gene.
Physical Characterization of Plasmid Clones
Plasmid DNA is extracted from E. coli cells using a standard alkali lysis
procedure, or other procedures known in the art (37). The DNA is digested
with a series of restriction endonucleases such as EcoRI, BamHI, HindIII,
PstI, EcoRV, and XbaI to establish digestion pattern of the clone and to
determine a size of the cloned DNA fragment. The physical map patterns for
the production clones were determined. The insert sizes for each clone are
calculated from this data and is summarized in Table 8.
TABLE 7
Cloned Esterase Candidates and Analysis
Recombinant
Specific
Activity Esterases Derivative
Active Activity in
Native in phage Identified in Primary Clone Plasmid
Plasmid Stabilized clone
# Strain lysate? Phage Lysate Name Name
Derivative U/mg
1 S1 + E001 lambdaTGE 1.1 pTGE1.1 +
1536
2 S1 + E001, E022 lambdaTGE 1.2 pTGE1.2 +
3 S1 + E001, E022 lambdaTGE 1.3 pTGE1.3 +
4 S1 + E001 lambdaTGE 1.4 pTGE1.4 +
5 S1 + E001 lambdaTGE 1.5 pTGE1.5 +
1489
6 S1 nt nt lambdaTGE 1.6 pTGE1.6 +
7 S1 nt nt lambdaTGE 1.7 pTGE1.7 +
8 S1 + E022 lambdaTGE 1.8 pTGE1.8 -
9 54 + E002 lambdaTGE 2.1 pTGE2.1 +
8300
10 54 + E023 lambdaTGE 2.2 pTGE2.2 nt
550
11 54 + E023 lambdaTGE 2.3 pTGE2.3 +
12 54 + E002 lambdaTGE 2.4 pTGE2.4 +
2530
13 54 + E002 lambdaTGE 2.8 pTGE2.8 -
14 50 + E003 lambdaTGE 3.1 pTGE3.1 -
15 50 + E003 lambdaTGE 3.2 pTGE3.2 +
2610
16 50 + E003 lambdaTGE 3.3 pTGE3.3 +
17 50 + E003 lambdaTGE 3.4 pTGE3.4 +
18 GP1 + E004 lambdaTGE 4.1 pTGE4.1 -
19 GP1 + E024 lambdaTGE 4.2 pTGE4.2 +
20 GP1 + E004 lambdaTGE 4.3 pTGE4.3 +
320
21 GP1 + E004 lambdaTGE 4.4 pTGE4.4 -
22 GP1 + E004 lambdaTGE 4.5 pTGE4.5 nt
23 GP1 + E004 lambdaTGE 4.6 pTGE4.6 +
490
24 C-1 + E005 lambdaTGE 5.1 pTGE5.1 -
25 C-1 + E025 lambdaTGE 5.2 pTGE5.2 +
26 C-1 + E005 lambdaTGE 5.3 pTGE5.3 +
984
27 C-1 - lambdaTGE 5.4 pTGE5.4 nt
28 C-1 + E005 lambdaTGE 5.5 pTGE5.5 nt
29 55 + E006 lambdaTGE 6.1 pTGE6.1 -
30 55 +/- E026 lambdaTGE 6.2 pTGE6.2 -
31 55 + E006 lambdaTGE 6.3 pTGE6.3 +
230
32 55 + E006 lambdaTGE 6.4 pTGE6.4 -
33 55 + E006 lambdaTGE 6.5 pTGE6.5 -
34 55 + E006 lambdaTGE 6.6 pTGE6.6 -
35 46 +- *** lambdaTGE 7.1 pTGE7.1 + 210
36 46 +- *** lambdaTGE 7.2 pTGE7.2 +
37 30 + E008 lambdaTGE 8.1 pTGE8.1 -
38 30 + E008 lambdaTGE 8.2 pTGE8.2 -
39 30 + E008 lambdaTGE 8.3 pTGE8.3 +
40 30 + E008 lambdaTGE 8.4 pTGE8.4 +
41 30 + E008 lambdaTGE 8.5 pTGE8.5 +
330
42 28 - lambdaTGE 9.1 pTGE9.1 +
43 28 - lambdaTGE 9.2 pTGE9.2 -
44 28 + E009 lambdaTGE 9.3 pTGE9.3 +
512
45 28 + E009 lambdaTGE 9.4 pTGE9.4 +
>270
46 28 + E009 lambdaTGE 9.5 pTGE9.5 -
47 28 + E009 lambdaTGE 9.6 pTGE9.6 +
48 28 + E009 lambdaTGE 9.7 pTGE9.7 +
49 29 - lambdaTGE 10.1 pTGE10.1 -
50 29 - lambdaTGE 10.2 pTGE10.2 -
51 29 + E010 lambdaTGE 10.3 pTGE10.3 +
546
52 29 - lambdaTGE 10.4 pTGE10.4 +
>600
53 29 + E010 lambdaTGE 10.5 pTGE10.5 +
54 29 + E010 lambdaTGE 10.6 pTGE10.6 -
55 29 - lambdaTGE 10.7 pTGE10.7 -
56 29 + E010 lambdaTGE 10.8 pTGE10.8 +
57 31 - lambdaTGE 11.1 pTGE11.1 +
58 31 - lambdaTGE 11.2 pTGE11.2 -
59 31 + E011 lambdaTGE 11.4 pTGE11.4 +
60 31 + E011 lambdaTGE 11.9 pTGE11.9 +
61 31 + E011 lambdaTGE 11.10 pTGE11.10 +
1052
62 31 - lambdaTGE 11.7 pTGE11.7 +
63 26b + lambdaTGE 12.1 pTGE12.1 +
64 26b + lambdaTGE 12.2 pTGE12.2 +
>600
65 26b + lambdaTGE 12.3 pTGE12.3 +
66 26b + lambdaTGE 12.4 pTGE12.4 +
67 26b + E029 lambdaTGE 12.5 pTGE12.5 -
68 26b + E029 lambdaTGE 12.6 pTGE12.6 -
69 27 + E013 lambdaTGE 13.1 pTGE13.1 +
70 27 + E013 lambdaTGE 13.2 pTGE13.2 +
428
71 27 + E013 lambdaTGE 13.3 pTGE13.3 +
33
72 27 + E013 lambdaTGE 13.4 pTGE13.4 +
73 34 - lambdaTGE 14.2 pTGE14.2 -
74 34 + E014 lambdaTGE 14.3 pTGE14.3 +
460
75 34 - lambdaTGE 14.4 pTGE14.4 -
76 34 + E014 lambdaTGE 14.5 pTGE14.5 +
>1200
77 34 + E027 lambdaTGE 14.6 pTGE14.6 +
>900
78 34 - lambdaTGE 14.7 pTGE14.7 +
79 34 + E014 lambdaTGE 14.8 pTGE14.8 -
80 34 + E014 lambdaTGE 14.9 pTGE14.9 +
81 62 + E015 lambdaTGE 15.1 pTGE15.1 +
82 62 + E015 lambdaTGE 15.2 pTGE15.2 +
83 62 + E015 lambdaTGE 15.3 pTGE15.3 +
84 62 + E015 lambdaTGE 15.4 pTGE15.4 +
85 62 + E015 lambdaTGE 15.5 pTGE15.5 +
86 62 + E015 lambdaTGE 15.6 pTGE15.6 +
87 62 + E015 lambdaTGE 15.7 pTGE15.7 +
89 62 + E015 lambdaTGE 15.9 pTGE15.9 +
4700
90 47 + E016 lambdaTGE 16.1 pTGE16.1 +
600
91 47 + lambdaTGE 16.2 pTGE16.2 +
92 47 + E016 lambdaTGE 16.3 pTGE16.3 +
>1200
93 47 + lambdaTGE 16.4 pTGE16.4 +
94 47 + E016 lambdaTGE 16.5 pTGE16.5 +
95 47 + lambdaTGE 16.6 pTGE16.6 +
96 47 + lambdaTGE 16.7 pTGE16.7 +
97 C-3 + lambdaTGE 18.1 pTGE18.1 +
nt
98 C-3 + lambdaTGE 18.2 pTGE18.2 -
99 4 + E019 lambdaTGE 19.1 pTGE19.1 +
>120
100 4 + E019 lambdaTGE 19.2 pTGE19.2 +
101 4 + E019 lambdaTGE 19.3 pTGE19.3 +
102 4 + E019 lambdaTGE 19.4 pTGE19.4 +
1960
103 4 + E019 lambdaTGE 19.5 pTGE19.5 -
104 4 + E019 lambdaTGE 19.6 pTGE19.6 +
105 7 - lambdaTGE 20.1 pTGE20.1 +
105 7 - lambdaTGE 20.2 pTGE20.2 +
106 7 + E020 lambdaTGE 20.3 pTGE20.3 +
2470
107 7 + E028 lambdaTGE 20.4 pTGE20.4 +
108 7 - lambdaTGE 20.5 pTGE20.5 +
109 7 + E020 lambdaTGE 20.6 pTGE20.6 +
110- 32 - lambdaTGE 21.1- pTGE21.1- +
104 21.5 21.5
105 32 + E017b lambdaTGE 21.6 pTGE21.6 +
106 32 + E017b lambdaTGE 21.8 pTGE21.8 +
930
107 32 + E017b lambdaTGE 21.9 pTGE21.9 +
***No protein detected by activity stain.
TABLE 8
Production Clone Data
Specific Activity
Approx. in Typical
Selected DNA Insert Recombinant
Production Production Recombinant Size.sup.1 Crude Extract.sup.2
Enzyme plasmid Strain Name (kb) (U/mg)
recE001 pTGE1.1 CE001 3.5 1,536
recE001.5 pTGE1.5 CE001.5 nt nt
recE002 pTGE2.1 CE002 2.5 8,300
recE003 pTGE3.2 CE003 4.1 2,610
recE004 pTGE4.6 CE004 3.4 490
recE005 pTGE5.3 CE005 1.9 984
recE006 pTGE6.3 CE006 6 230
recE007 pTGE7.1 CE007 3.7 210
recE008 pTGE8.5 CE008 3.2 330
recE009 pTGE9.4 CE009 4.5 270
recE010 pTGE10.3 CE010 2.5 546
recE011 pTGE11.10 CE011 2.4 1,052
recE029 pTGE12.2 CE029 4.2 600
recE013 pTGE13.2 CE013 2.2 428
recE014 pTGE14.3 CE014 2.5 460
recE015 pTGE15.9 CE015 3.5 4,700
recE016 pTGE16.1 CE016 2 600
recE016.3 pTGE16.3 CE016.3 1.8 1,200
recE017b pTGE21.8 CE017b 3.8 930
recE019 pTGE19.4 CE019 3.7 1,960
recE020 pTGE20.3 CE020 2.7 2,470
recE022 pTGE1.8 CE022 nt nt
recE023 pTGE2.2 CE023 3.7 550
recE024 pTGE4.2 CE024 nt nt
recE025 pTGE5.2 CE025 nt nt
recE027 pTGE14.6 CE027 2.6 900
recE028 pTGE20.4 CE028 2.5 nt
.sup.1 Insert sizes are estimated from the agarose gel. The estimated
insert size is based on a vector size of 4.5 kb and the accuracy which
could be achieved analyzing each of the six digestion patterns.
.sup.2 Specific activity is calculated as the amount of p-nitrophenol
produced in micromoles per minute per milligram of total protein as
described in Example 2. The numbers reported here are from a typical
production batch and may vary.
Generation of the tag sequences for PCR identification of esterase
containing inserts
The DNA sequences of the ends of the insert fragment carrying esterase
genes can be determined by sequencing the ends of the inserts using
standard T7 and S6 primers to produce unique tags of the cloned DNA.
Sequence analysis can be carried out to design PCR primers which can
uniquely amplify the DNA inserts from both the clones and the host
organisms. These tags can be potentially used to generate this DNA
fragment from the chromosome of the studied organisms using PCR technique.
Screening of the Cosmid library with an oligonucleotide probe
For cloning of enzymes which cannot be cloned by activity, other methods
are used. A degenerative probe is prepared to the N-terminal sequence of
the protein and hybridized to plaques from the recombinant phage bank.
Alternatively, degenerate PCR amplification probes can be made using the
N-terminal sequence or sequences obtained from the n-termini of internal
protein fragments which have been obtained after proteolytic digestion of
the enzyme. Using these sequences, a probe can be made from an amplified
region between the N-terminus and an internal fragment or between two
internal fragment sequences to identify a clone carrying the DNA encoding
for the enzyme of interest.
EXAMPLE 18
Overproduction and Overexpression of Esterases
Production of recombinant esterase
The production strains used are listed in Table 8. Cloned enzymes are
produced from E. coli. strain XLOLR. Alternatively, any suitable E. coli
host may be used, including but not limited to HB101, C600, TG1 and
XL1-Blue.
Several media can be used to produce cloned esterases. LB (10 gm/l
tryptone, 5 gm/l yeast extract and 10 gm/l NaCl) and Terrific Broth (12
gm/l tryptone, 24 gm/l yeast extract and 4 ml/l glycerol supplemented with
100 ml of a sterile solution of 0.17 M KH.sub.2 PO.sub.4, 0.72 M K.sub.2
HPO.sub.4 after autoclaving) have been tested and the results from optimal
growth conditions for the production strains listed in Table 9 below. Each
media is supplemented with 10-50 .mu.g/ml kanamycin.
Optimal production media depends on a number of factors, including media
cost and specific activity of the produced proteins. TB media is a richer
media and therefore more expensive. For instance, in the case of CE009,
while more total units are produced in a single fermentation run, not
enough is produced to justify the higher cost of the media. In addition,
the specific activity is higher for the LB media preparation.
Fermentation production is run in 17 L Fermentors (15 L working volume/LH
Fermentation) at 30.degree. C., 600 RPM, and 0.5 vvm air flow. The seed
train is established as follows. A loopful of a frozen production culture
is used to inoculate 50 ml of production media in a 250 ml Erlenmeyer
flask. The flask is incubated at 30.degree. C. for two days (250 RPM) and
then used to inoculate a 1 liter flask with 250 ml of production media.
This flask is incubated 1 day at 30.degree. C. and 250 RPM. The 1 liter
flask is used to inoculate the fermentor.
Production of substantially purified preparations from a cell paste of
strains producing the recombinant enzymes are carried out similar to the
methods described in Example 4 and the specific protocols described in
Examples 14-34 for the native proteins.
TABLE 9
Preferred media for Strains CE001-CE010
LB TB
Specific Total Specific Total
Current
Activity Cell mass Total Activity Cell mass Total
Growth media
Strain (U/mg) (g) Units (U/mg) (g) Units of
choice*
CE001 213 0.41 4500 138 0.84 6725 TB
CE002 98 0.52 1625 101 0.93 4575 TB
CE003 272 0.42 4200 22 0.87 1025 LB
CE004 208 0.47 3650 28 0.90 1350 LB
CE005 123 0.40 3675 125 1.00 7600 TB
CE006 85 0.42 2125 71 0.62 2175 LB
CE007 9 0.39 225 19 0.75 500 TB
CE008 71 0.51 2775 45 0.80 2350 LB
CE009 109 0.42 2650 74 0.81 3050 LB
CE010 418 0.42 2200 225 0.95 8375 TB
*Given current media costs
Optimization of esterase production
Further optimization of esterase production is performed by media studies
in shake flasks followed by further optimization at the 1 liter to 20
liter scale. Depending on the enzyme, final fermentation conditions can
involve either a fed-batch or continuous fermentation process. Since the
esterase activity being analyzed is intracellular, the use of a clear or
defined media such as TT media is necessary. Organisms of interest are
grown and cell pellets are collected by centrifugation. Pellets are
disrupted by sonication and enzymes can be purified using the standard
techniques of ion exchange and gel permeation chromatography described in
Examples 3 and 4. Growth conditions including media composition, pH, and
temperature are optimized at the small scale (ie. shake flasks, and 1
liter fermentors) to give the highest cell density while retaining the
highest amount of enzyme.
Isolation of High-production mutants
Several simple mutagenesis schemes are used to try and isolate
high-producing mutants of the different activities of interest. These
include mutagenesis with uv-light or chemical mutagens such as
ethylmethane sulfanoate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG). The cells are treated with varying concentrations of the mutagen
(or varying exposure times with uv light) according to methods described
in Miller (38). Optimal concentrations of the different mutagens with
different organisms vary. In general, killing concentrations allowing 80%
survival for EMS, approximately 50% survival for MNNG, or 10-50% survival
for uv light are desired. Mutagenized cultures are then grown up, allowing
the mutagen to wash out and plated onto solid media.
Mutants are identified by applying an esterase plate screen to the cells.
For example with an esterase screen, an agar overlay containing a
colorimetric or fluorogenic substrate such as
5-bromo-4-chloro-3-indolyl-acetate or 4-methyulumbelliferyl acetate will
be applied. Colonies which show a significant increase in activity by
hydrolysis of the substrate will be identified.
Candidate mutants are then analyzed by native polyacrylamide gel
electrophoresis and compared to the parental strain. Standard assay
methods described in Example 2 or the rapid esterase/lipase screen
described in Example 12 can then be applied to identify any differences in
amounts of enzyme activity. If a production level increase is large an
increased band on either a Native or SDS polyacrylamide gel after
coomassie staining may be seen. Strains with multiple activities can also
be differentiated in this way, verifying that the increase is in the
enzyme of interest. It is then confirmed that the mutants have unaltered
kinetic and substrate properties as the parental enzyme. The majority of
mutations identified by this approach are expression mutations which can
be isolated in either a promoter region, repressor molecule, or other
controlling element. Most mutations in the enzyme structural genes will
likely inactivate the enzyme, however, an enhanced activity may also be
isolated. If it is apparent that the mutation increases the activity of
the desired protein band but not the intensity of the band on a coomassie
stained gel, the mutant is recharacterized to determine if it is a more
efficient biocatalyst.
EXAMPLE 19
Esterase Screening Kit
A large subset of enzymes can be packaged into an easy to use screening kit
to rapidly analyze a large number of enzymes at once. The kits are
formulated to eliminate as many potential errors as possible and each
enzyme is provided in a lyophilized form if possible near its optimal
buffer and reaction conditions.
Many different formats for the kit are possible, from a series of glass
vials, to varying size microtiter plates constructed of different plastic
materials. The microtiter plate is favored because of its ease of handling
and manipulating. Most microtiter plates are made of polystyrene however,
which will not stand up to most organic solvents. For experiments which
utilize aqueous solvent, the polystyrene is not a problem. Other more
tolerant plastics such as polypropylene are available and are ideal for
the kit. Large size 24-well microtiter plates which allow 3 ml of sample
to be assayed (allowing enough sample for multiple TLC or HPLC analysis)
have been developed. Other formats may also be useful for different
applications.
Each kit is prepared by addition of a stir bar, buffer (0.1M Na phosphate
pH 7.0) and 1 U of each enzyme to each well of a 24 well polypropylene
tray (Tomtec). Enzymes are aliquotted into each well or vial in set
amounts so that it can be assured that an equal amount of activity is
provided for comparison. The entire kit is then lyophilized, sealed with
heat seal foil (3M) and labeled. Separate experiments found that there was
no significant loss in enzyme activity when proteins were lyophilized in
the kit trays as suggested by earlier experiments comparing glass to
plastic. In addition to enzymes, each kit contains four control wells that
are composed of buffers at pH's from 6-9 since it was found that some of
the substrates tested tend to be unstable in buffered solutions which can
confuse positive results with autohydrolysis. The rest of the kit is
composed of an instruction manual, a data sheet, a sample preparation vial
a glass eye dropper and a plastic eye dropper. The kit is formulated in
such a way that only solvent and substrate need be added to each well. The
rapid-screen indicator dye method described in Example 12 can also be
included in each well or vial. This makes a preliminary qualitative
determination of enzyme effectiveness simple and fast.
EXAMPLE 20
Cloning and Characterization of Recombinant Proteins
The cloning and characterization of recombinant proteins from strain
isolates which produced the native isolated protein (as listed in Table 1)
was carried out as described in Example 37. Lambda expression vectors were
isolated as described above (specific named isolates are shown in Table
7). E. coli clones harboring the excised hybrid phage-plasmids were
derived as summarized in Table 7, and were finally selected for esterase
activity by subsequent screening, which after 3 rounds of stabilizing
procedure was calculated to approximate units of activity per mg of total
cell protein obtained. Esterase activity stain gel used to screen positive
phage library candidates for the recombinant proteins allowed the
identification of alternative recombinant proteins as well. Production of
the recombinant protein is carried out as described above, using TB for
the media and purifying the enzyme as described for the native
(nonrecombinant) protein in Example 4.
EXAMPLE 21
Sequencing of Recombinant Proteins
The isolation and cloning of the genes encoding for the enzymes of the
instant invention results in DNA segments in which an open reading frame
(ORF) may be found which corresponds to translated protein amino acid
sequence. Sequencing of the DNA inserts which contain the corresponding
nucleic acid sequence which encode for the protein enzymes can be
conducted by the usual methods, either manually or using automated
apparatus.
Once obtained, analysis of the nucleic acid sequence can reveal the
presence of alternative start codons, a phenomenon recognized in the art,
however the encoded protein enzyme will comprise at minimum a core protein
ORF. FIG. 6A is an isolated nucleic acid sequence, and translated amino
acid sequence which correspond to E001 (SEQ ID NO.:1 and SEQ ID NO.:2)
enzyme ORF, alternative start codons are underlined. FIG. 6B is an
isolated nucleic acid sequence, and translated amino acid sequence which
correspond to E009 (SEQ ID NO.:3 and SEQ ID NO.:4) enzyme ORF, alternative
start codons are underlined. FIG. 6C is the cloned isolated nucleic acid
sequence which contains the E011 (SEQ ID NO.:5 and SEQ ID NO.:6) ORF,
alternative start codons are underlined. FIG. 6D is the cloned isolated
nucleic acid sequence which contains the E101 (SEQ ID NO.:7 and SEQ ID
NO.:8) ORF, alternative start codons are underlined. FIG. 6E is the cloned
isolated nucleic acid sequence which contains the E019 (SEQ ID NO.:9 and
SEQ ID NO.:10) ORF, alternative start codons are underlined. FIG. 6F is
the cloned isolated nucleic acid sequence which contains the E005 (SEQ ID
NO.:11 and SEQ ID NO.:12) ORF, alternative start codons are underlined.
FIG. 6G is the cloned isolated nucleic acid sequence which contains the
E004 (SEQ ID NO.:13 and SEQ ID NO.:14) ORF, alternative start codons are
underlined. FIG. 6H is the cloned isolated nucleic acid sequence which
contains the E006 (SEQ ID NO.:15 and SEQ ID NO.:16) ORF, alternative start
codons are underlined. FIG. 6I is the cloned isolated nucleic acid
sequence which contains the E008 (SEQ ID NO.:17 and SEQ ID NO.:18) ORF,
alternative start codons are underlined. FIG. 6J is the cloned isolated
nucleic acid sequence which contains the E010 (SEQ ID NO.:19 and SEQ ID
NO.:20) ORF, alternative start codons are underlined. FIG. 6K is the
cloned isolated nucleic acid sequence which contains the E013 (SEQ ID
NO.:21 and SEQ ID NO.:22) ORF, alternative start codons are underlined.
FIG. 6L is the cloned isolated nucleic acid sequence which contains the
E015 (SEQ ID NO.:23 and SEQ ID NO.:24) ORF, alternative start codons are
underlined. FIG. 6M is the cloned isolated nucleic acid sequence which
contains the E016 (SEQ ID NO.:25 and SEQ ID NO.:26) ORF, alternative start
codons are underlined. FIG. 6N is the cloned isolated nucleic acid
sequence which contains the E017 (SEQ ID NO.:27 and SEQ ID NO.:28) ORF,
alternative start codons are underlined. FIG. 6O is the cloned isolated
nucleic acid sequence which contains the E020 (SEQ ID NO.:29 and SEQ ID
NO.:30) ORF, alternative start codons are underlined. FIG. 6P is the
cloned isolated nucleic acid sequence which contains the E027 (SEQ ID
NO.:31 and SEQ ID NO.:32) ORF, alternative start codons are underlined.
FIG. 6Q (SEQ ID NO.:33) contains the nucleic acid sequence of the 5' end,
and FIG. 6R (SEQ ID NO.:34) contains the 3' end of the insert which
contains the E003. FIG. 6S (SEQ ID NO.:35) contains the nucleic acid
sequence of the 5' end, and FIG. 6T (SEQ ID NO.:36) contains the 3' end of
the insert which contains the E004 ORF. FIG. 6U (SEQ ID NO.:37) contains
the nucleic acid sequence of the 3' end of the insert which contains the
E014 ORF. These nucleic acid sequences allow one of ordinary skill in the
art, practicing routine methods to complete characterization of the full
length nucleic acid sequence of the insert, the detection of clones via
hybridization, and the creation of amplification primers for detecting,
amplifying and generating full length homologous genes.
TABLE 10
ThermoCat .TM. E001-E020 Spec comparison
Specific Temperature pH
Half Life (hours)
Biocatalyst Activity MW Opt. Useful Range Opt.
50% Range 40.degree. C. 60.degree. C.
E001 0.5 u/mg 22 kDal 45.degree. C. RT-55.degree. C. 7.5
broad +++ 34
E002 1.0 u/mg 28 kDal 45.degree. C. RT-60.degree. C. 7.0
broad +++ 30
E003 0.5 u/mg 28 kDal 45.degree. C. RT-60.degree. C. 7.0
broad +++ 60
E004 0.6 u/mg 36 kDal 45.degree. C. RT-60.degree. C. 6.5
<6.0-8.0 +++ 10
E005 6.7 u/mg 28 kDal 45.degree. C. RT-60.degree. C. 7.0
broad +++ 15
E006 3.6 u/mg 36 kDal 45.degree. C. RT-60.degree. C.
6.5-7.0 broad +++ 30
E007 2.7 u/mg 28 kDal 35.degree. C. RT-60.degree. C. 7.0
<6.0-8.0 >480 90
E008 1.5 u/mg 28 kDal 40.degree. C. RT-55.degree. C.
6.5-7.0 <6.0-8.0 50 <1
E009 1.3 u/mg 36 kDal 45.degree. C. RT-50.degree. C.
6.5-7.0 <6.0-8.0 +++ <1
E010 4.9 u/mg 46 kDal 45.degree. C. RT-55.degree. C. 6.5
<6.0-8.0 +++ <1
E011 6.2 u/mg 36 kDal 45.degree. C. RT-60.degree. C.
6.5-7.0 <6.0-8.0 +++ 4
E012 10.7 u/mg 28 kDal 45.degree. C. RT-60.degree. C. <=6.0
<6.0-7.5 +++ 240
E013 5.3 u/mg 36 kDal 45.degree. C. RT-60.degree. C. 7.0
<6.0-8.0 >480 6
E014 0.9 u/mg 36 kDal 45.degree. C. RT-50.degree. C. 7.0
<6.0-8.0 +++ <1
E015 3.0 u/mg 36 kDal 45.degree. C. RT-60.degree. C. >9.0
7.5->9.0 +++ 6
E016 1.2 u/mg 28 kDal 45.degree. C. RT-60.degree. C. nd
nd +++ 240
E017b 0.4 u/mg 36 kDal 40.degree. C. RT-50.degree. C. >9.0
7.5->9.0 +++ 4
E018 0.2 u/mg nd nd nd nd nd
120 30
E019 0.9 u/mg 30 kDal 45.degree. C. RT-60.degree. C. >9.0
broad nd 25
E020 3.9 u/mg 28 kDal 45.degree. C. RT-60.degree. C. broad
broad +++ 12
*broad pH range refers to >50% activity through all pH tested (6.0-8.5)
EXAMPLE 22
Ester Chain Length Specificity Characterization
The enzymes of the instant invention can be further characterized by
testing for enzymatic specificty for substrate esters of different chain
length. Such assays can be conducted using the methods described above,
selecting the appropriate substrates. FIG. 7 depicts the result of
colormetric esterase activity assays of the various enzymes. The graphed
data was obtained where the reaction conditions were estimated to be
approximately 0.1 U/l ml reaction, with 500 ug/ml substrate, where 1 Unit
(U) is calculated for each enzyme stock preparation in relation to
esterase activity where 1 Unit is the amount of enzyme needed to hydrolize
approximately 1 umol of p-nitrophenyl proprionate per minute. The data is
reported as approximate maximum OD.sub.410 nm during incubation.
FIG. 7A graphs data using the substrate bis-p-nitrophenyl-carbonate. The
highest activity was found with enzyme E019, which showed an OD.sub.410 nm
of 0.30 after 4 hours incubation. FIG. 7B graphs data using the substrate
p-nitrophenyl-acetate. The highest activity was found with enzyme E020,
which showed an OD.sub.410 nm of 3.571 after 400 seconds incubation. FIG.
7C graphs data using the substrate bis-p-nitrophenyl-propionate. The
highest activity was found with enzyme E003, which showed an OD.sub.410 nm
of 1.4 after 600 seconds incubation. FIG. 7D graphs data using the
substrate bis-p-nitrophenyl-butyrate. The highest activity was found with
enzyme E020, which showed an OD.sub.410 nm of 1.19 after 160 seconds
incubation. FIG. 7E graphs data using the substrate
bis-p-nitrophenyl-caproate. The highest activity was found with enzyme
E009, which showed an OD.sub.410 nm of 0.37 after 560 seconds incubation.
FIG. 7F graphs data using the substrate bis-p-nitrophenyl-caprylate. The
highest activity was found with enzyme E003, which showed an OD.sub.410 nm
of 0.07 after 360 seconds incubation. FIG. 7G graphs data using the
substrate bis-p-nitrophenyl-laurate. The highest activity was found with
enzyme E016, which showed an OD.sub.410 nm of 0.11 after 480 seconds
incubation.
EXAMPLE 23
pH Dependent Assay for Entantiomer Esterase Specificity
The enzymes of the invention can be further characterized by testing for
enzymatic specificity for specific entantiomer substrate esters of
different chiral structure. Such assays can be performed using the methods
described above, selecting the appropriate substrate. The results of
screening are depicted in FIG. 8. FIG. 8A summarizes the results of
colorometric esterase activity assays for entaniomer specificity. FIG. 8B
depicts quantitative colorometric assay data results in terms of minutes
required for detectable color change, indicating pH change. The numbers
report time in minutes following addition of enzyme. NH indicates no
hydrolysis was detected after 3 days, and o/n indicates no hydrolysis
after overnight incubation (approximately 6-15 hours). Substrates 1, 2, 4,
6, 8, and 9 were dissolved to a concentration of 40 mM in a 25 mM KPi
buffer, pH=7.4, containing .about.0.005% of bromothymol blue. Substrates
3, 5 and 7 were dissolved to a concentration of 10 mM in a 5 mM KPi
buffer, pH=7.4, containing .about.0.005% of bromothymol blue and up to 10%
MeCN as cosolvent. The esterases tested were added in the amount of 1 U
per well, as determined by hydrolysis of PNP-propionate. The control
reaction was the substrate solution, with no added enzyme.
EXAMPLE 24
Characterization of Enzyme Activity Against Para-nitroanilide Compounds
The enzymes of the invention can be further characterized by testing for
enzymatic specificity for alternative substrates which are similar to
esters. Such assays can be performed using the methods described above,
selecting the appropriate substrates. The enzymes of the invention were
characterized against the anilides and esters listed below and the results
depicted in FIG. 9. The assays were performed according to the general
formula:
##STR7##
Test reactions were run in microtiter plates with each reaction in a total
volume of about 100 ul. Each reaction consisted of about 75 ul of pH7.0
phosphate buffer, 5 ul of 5 mM substrate, and 20 ul of enzyme adjusted to
50 U/ml (where I U is approximatly the amount needed to hydrolize 1 uM of
p-nitrophenyl-propionate in 1 minute). The final reaction mixture
contained about 1 U enzyme and 0.25 mM substrate in each well. The
reactions were incubated for about 2.5 hours at 37C. Control reactions,
lacking enzyme, were run in adjacent wells. A control containing no
substrate was also run on each plate. Following incubation, the plates
were read at 405 nm in a BIORAD Model 3550 microplate reader. Values of
the controls were subtracted from the experimental well values to
determine net activity.
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